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Molybdoenzyme that catalyzes the anaerobic hydroxylation of a tertiary
carbon atom in the side chain of cholesterol Juri Dermer and Georg Fuchs
From Lehrstuhl Mikrobiologie, Fakultät Biologie, Universität Freiburg,
Schänzlestr. 1, D-79104 Freiburg, Germany
Running title: Steroid C25 dehydrogenase
Address correspondence to: Georg Fuchs, Mailing address: Mikrobiologie, Fakultät Biologie,
Schänzlestr. 1, D-79104 Freiburg, Germany; Phone: 49-761-2032608; Fax: 49-761-2032626; E-mail:
Keyword: molybdenum hydroxylase; anaerobic steroid metabolism
Background: Cholesterol degradation is
challenging due to its complex structure and low
water solubility.
Results: C25 dehydrogenase is a novel
molybdenum/iron-sulfur/heme-containing
enzyme that hydroxylates the tertiary C25 of the
steroid side chain.
Conclusion: C25 dehydrogenase and related
enzymes identified in the genome of
Sterolibacterium denitrificans replace
oxygenases in anaerobic and even aerobic steroid
metabolism.
Significance: O2-independent hydroxylations by
molybdoenzymes probably represent a general
strategy to activate steroid substrates
anaerobically.
SUMMARY
Cholesterol is a ubiquitous hydrocarbon
compound that can serve as substrate for
microbial growth. This steroid and related
cyclic compounds are recalcitrant owing to
their low solubility in water, complex ring
structure, the presence of quaternary carbon
atoms, and the low number of functional
groups. Aerobic metabolism therefore makes
use of reactive molecular oxygen as co-
substrate of oxygenases to hydroxylate and
cleave the sterane ring system. Consequently,
anaerobic metabolism must substitute oxyge-
nase catalyzed steps by O2-independent
hydroxylases. Here we show that one of the
initial reactions of anaerobic cholesterol
metabolism in the betaproteobacterium
Sterolibacterium denitrificans is catalyzed by
an unprecedented enzyme that hydroxylates
the tertiary C25 atom of the side chain
without molecular oxygen forming a tertiary
alcohol. This steroid C25 dehydrogenase
belongs to the dimethylsulfoxide dehydro-
genase molybdoenzyme family, the closest
relative being ethylbenzene dehydrogenase. It
is a heterotrimer, which is probably located at
the periplasmic side of the membrane and
contains 1 molybdenum cofactor, 5 [Fe-S]-
clusters, and 1 heme b. The draft genome of
the organism contains several genes coding
for related enzymes that likely replace
oxygenases in steroid metabolism.
INTRODUCTION
Cholesterol is one of the most abundant
and ubiquitous steroids. It serves as an essential
constituent of eukaryotic membranes and acts as
a precursor molecule for the biosynthesis of
steroid hormones, oxysterols, and bile acids.
Steroids are also formed by plants (1) and some
prokaryotes (2), but their degradation is mostly
limited to microorganisms. Microbial degrada-
tion of the omnipresent steroids like cholesterol
and the analogous plant and fungal steroids
(stigmasterol, β-sitosterol, ergosterol) is an
important issue of the global carbon cycle.
Furthermore, microbial transformation of steroid
molecules is an essential part of the biotechno-
logical production of steroid drugs (3).
Degradation of cholesterol is challenging
because of its low solubility in water (95 µg l-1
,
0.25 µM) and particularly because of its complex
chemical structure harboring quaternary carbon
atoms and a low number of functional groups.
Owing to this recalcitrance, cholesterol and
related steroids are used as biological markers in
studying the biological origin and fate of organic
compounds in geological records (4). Never-
theless, the ability to grow on cholesterol as a
sole carbon source is widespread in the microbial
world. Nocardia, Mycobacterium, Pseudomonas,
Arthrobacter, and Rhodococcus species are well
known cholesterol degrading aerobes (5), some
of those bacteria being used in biotechnology
(3). Under aerobic conditions, mono- and dioxy-
http://www.jbc.org/cgi/doi/10.1074/jbc.M112.407304The latest version is at JBC Papers in Press. Published on September 1, 2012 as Manuscript M112.407304
Copyright 2012 by The American Society for Biochemistry and Molecular Biology, Inc.
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genases are involved in cholesterol metabolism.
They catalyze crucial hydroxylation steps that
initiate degradation of the side chain and bring
about the cleavage of the sterane ring (5).
Obviously, the anoxic metabolism needs
to substitute all essential O2-dependent steps by
an oxygen-independent strategy to overcome the
chemical recalcitrance of the molecule. This
challenge prompted us to study the anoxic
metabolism of cholesterol in the β-proteo-
bacterium Sterolibacterium denitrificans (6), one
of two strains that are known to grow an-
aerobically (but also aerobically) on cholesterol
as sole carbon source (7). In a previous work, the
initial steps of this anoxic metabolism were
identified (8-10) (Fig. 1). Cholesterol is
converted to cholest-4-en-3-one by a
bifunctional cholesterol dehydroge-
nase/isomerase. Cholest-4-en-3-one is further
oxidized to cholesta-1,4-dien-3-one catalyzed by
cholest-4-en-3-one-∆1-dehydrogenase. Both
transformations show analogy to the aerobic
cholesterol metabolism. Recently, similar
reactions were identified in anaerobic degra-
dation of testosterone in Steroidobacter
denitrificans (11).
Intriguingly, the following degradation
of cholesterol involves an unprecedented
anaerobic hydroxylation of the tertiary C25 atom
of the side chain resulting in the formation of a
tertiary alcohol. This reaction fundamentally
differs from the O2-dependent hydroxylations
that occur in aerobic steroid metabolism. It is
well known that in anaerobic pathways
molybdenum-containing hydroxylases can be
regarded as a counterpart to oxygenases
functioning in aerobic metabolism. They use
water as source of the oxygen atom incorporated
into the product and require an electron acceptor;
in contrast, oxygenases use molecular oxygen as
source of oxygen and many require an electron
donor.
A paradigm for such an anaerobic
hydroxylase acting on a hydrocarbon side chain
is ethylbenzene dehydrogenase (12), and the
analogous cholesterol C25 hydroxylation was
proposed to be catalyzed by a similar
molybdenum enzyme (10). Ethylbenzene
dehydrogenase belongs to type II molybdenum
containing enzymes of the dimethylsulfoxide
(DMSO) reductase family and catalyzes the
hydroxylation of ethylbenzene to (S)-phenyl-
ethanol.
Here we purified and characterized
steroid C25 dehydrogenase (briefly C25 de-
hydrogenase). The molybdenum containing en-
zyme is membrane associated and genes coding
for the subunits of the heterotrimeric enzyme
were identified in the draft genome of the
organism. Furthermore, analysis of the genome
revealed the presence of at least seven further
proteins with high similarity to the molybdenum-
containing large subunit of ethylbenzene de-
hydrogenase and C25 dehydrogenase (hydroxyl-
lase). In contrast, no gene coding for an
oxygenase acting on steroids was found in the
genome. It appears indeed that several
molybdenum dependent anaerobic hydroxylases
take over the role of oxygenases in anaerobic
metabolism of steroids. In this organism, the
anaerobic strategy is even used for the aerobic
metabolism of steroids.
EXPERIMENTAL PROCEDURES
Materials and Bacterial Strain - The
chemicals used were of analytical grade and
were purchased from Sigma-Aldrich
(Heidelberg, Germany), Merck (Darmstadt,
Germany), Roth (Karlsruhe, Germany), or Santa
Cruz Biotechnology (Heidelberg, Germany).
Sterolibacterium denitrificans Chol-1ST (DSMZ
13999) was obtained from the Deutsche
Sammlung für Mikroorganismen und
Zellkulturen (Braunschweig, Germany).
Materials and equipment for protein purification
were obtained from Sigma-Aldrich (Heidelberg,
Germany) and GE Healthcare (München,
Germany).
Bacterial Cultures and Growth
Condition - S. denitrificans was grown on
cholesterol at 30 °C under oxic as well as under
anoxic, denitrifying conditions as described
(6,9). Cells were harvested by centrifugation in
the exponential growth phase at an optical
density (OD578 nm) of 1.0 to 1.6 (optical path 1
cm) and then stored at -70 °C. Large scale
fermenter cultures (200 liters) were set up as
described previously (10).
Preparation of Cell Extracts - Cell
extracts were prepared at 4 °C under anoxic
conditions. Frozen cells were suspended in two
volumes of 20 mM Tris/H3PO4 buffer (pH 7.0)
containing 0.1 mg of DNase I ml-1
. Cells were
broken by passing the cell suspension through a
French pressure cell (American Instruments,
Silver Spring, MD) twice at 137 MPa. The cell
lysate was fractionated by two steps of
centrifugation: at first, lysate was centrifuged for
30 min at 10,000 x g to get rid of the debris,
unbroken cells, and residual undissolved
cholesterol. Then, the supernatant (crude cell
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extract) was centrifuged at 150,000 x g for 2 h to
separate soluble proteins from membrane-bound
proteins. Extracts of aerobically grown cells
were prepared under oxic conditions. Separation of Subcellular Compart-
ments - Steroid C25 dehydrogenase localization
was studied under anoxic conditions. 50 ml of an
exponentionally growing culture (OD578 1) were
harvested by centrifugation at 2,800 x g for 15
min. To lyse the cells gently, the cell pellet was
resuspended in 10 ml buffer containing 20 mM
Tris/H3PO4 (pH 7.0), 1 M KCl, 10 mg lysozyme
ml-1
(100,000 Units mg-1
), 5 mg polymyxin B ml-
1, 1 mM dithioerythritol (DTE), and 0.1 mg
DNase I. The cell slurry was incubated on ice for
two hours and then centrifuged at 150.000 x g
for two hours. Supernatant containing the soluble
cell proteins was separated from the pellet
containing the membrane proteins. The pellet
was then resuspended in buffer containing 20
mM Tris/H3PO4 (pH 7.0) and 1 % Tween 20
(w/v). Both fractions were assayed for C25
dehydrogenase, cholest-4-en-3-one-∆1-
dehydrogenase, and malate dehydrogenase
activities.
To separate cytoplasmic membrane from
outer membrane, a sucrose gradient
centrifugation was carried out. 40 ml of an
exponentionally growing culture (OD578 1.4)
were harvested by centrifugation at 2,800 x g for
15 min. Cells were resuspended in two volumes
of buffer (20 mM Tris/H3PO4 (pH 7.0), 0.1 mg
DNase I ml-1
), and disrupted by passing through
the French press cell. The cell extract was
centrifuged at 10,000 x g for 20 min. The
supernatant (1.5 ml) was layered on top of a
sucrose bed, consisting of 0.6 M (8 ml), 0.9 M (8
ml), and 1.75 M (8 ml) sucrose in 20 mM
Tris/H3PO4 buffer (pH 7.0), and centrifuged at
38,000 x rpm (100,000 x g) for 16 h (rotor 60 Ti,
Beckman). Three fractions were carefully
collected by removal of material from top of the
gradient: (i) the soluble protein fraction (0.6 M
sucrose), (ii) the fraction containing the
cytoplasmic membrane (distinct layer between
0.6 M and 0.9 M sucrose) and (iii) the outer
membrane and unbroken cells (1.75 M sucrose).
The collected fractions were assayed for C25
dehydrogenase and malate dehydrogenase
activities.
In Vitro Assays - An HPLC-based C25
dehydrogenase assay was routinely performed
anaerobically under a nitrogen gas phase at 30
°C. The assay mixture (0.3 ml) contained 20 %
(w/v) (2-hydroxypropyl)-β-cyclodextrin, 100
mM potassium phosphate buffer (pH 7.5), 0.5
mM cholest-4-en-3-one (from 52 mM stock
dissolved in 1,4-dioxane), and 5 mM
K3[Fe(CN)6]. The reaction was started by
addition of enzyme, and the mixture was shaken
at 700 rpm. Samples of 80 µl were taken at
intervals, and the reaction was stopped by
addition of 20 µl of 25 % HCl. Samples were
centrifuged for 10 min at 20,000 x g, and 80 µl
were analyzed for substrate and products by
reverse-phase (RP) HPLC and UV detection at
240 nm . Note, that the calculated hydroxylation
rate based on UV detection of the product at 240
nm can be affected by further reactions of the
product in cell extract, which might result in
disappearance of the conjugated double bond
system and therefore in absorption decrease at
240 nm. When detergent was used instead of
cyclodextrin, the reaction mixture (0.3 ml)
contained 0.1 M potassium phosphate buffer (pH
7.0), 10 mM K3[Fe(CN)6], 2.6 mM cholest-4-en-
3-one, and 0.5 % detergent (e.g. Tween 20,
Triton X-100). The reaction was started by
addition of enzyme, and the mixture was shaken
at 700 rpm. The reaction was stopped by
extraction with two volumes of ethyl acetate.
The ethyl acetate soluble fraction was
concentrated under vacuum by vacuum
concentrator (Bachofer, Reutlingen, Germany)
and analysed by HPLC. For assaying C25
dehydrogenase in whole cells, 2 M sucrose was
added to the assay mixture to avoid cell lysis.
Alternatively, purified C25
dehydrogenase was assayed spectro-
photometrically with ferricenium tetrafluoro-
borate (FcBF4) as electron acceptor. The assay
mixture (0.5 ml) contained 28 % (w/v)
cyclodextrin, 100 mM potassium phosphate
buffer (pH 7.5), 0.5 mM cholest-4-en-3-one, and
0.5 mM FcBF4. The reaction was started by
addition of enzyme, and the decrease of
absorption caused by reduction of the
ferricenium ion was followed at 300 nm (∆ε =
3,587 M-1
cm-1
). To couple substrate
hydroxylation to cytochrome c reduction,
oxidized bovine heart cytochrome c was used in
a 40-fold molar excess to C25 dehydrogenase,
instead of ferricenium tetrafluoroborate. The
reaction was started by the addition of 0.5 mM
cholest-4-en-3-one to the reaction mixture and
the reduction of cytochrome c was followed at
550 nm ( ∆ε = 20,000 M-1
cm-1
) (13).
Malate dehydrogenase activity was measured at
30 °C in a reaction mixture (0.5 ml) containing
100 mM potassium phosphate buffer (pH 7.5),
0.25 mM NADH, and 0.2 mM oxaloacetate.
Oxidation of NADH was followed
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spectrophotometrically at 365 nm (∆ε = 3,400 M-
1 cm
-1) with a filter spectrophotometer.
Cholest-4-en-3-one-∆1-dehydrogenase was
assayed at 30 °C in 0.1 M potassium phosphate
buffer (pH 7.5) containing 5 mM 2,6-
dichlorophenol-indophenol, 0.5 mM cholest-4-
en-3-one, and 20 % (w/v) cyclodextrin. The
reaction was started by addition of enzyme
solution and the mixture (0.3 ml) was shaken at
700 rpm aerobically. Samples of 80 µl were
taken at intervals and the reaction was stopped
by addition of 20 µl of 25 % HCl. Samples were
centrifuged for 10 min at 20,000 x g, and 80 µl
were analyzed by reverse-phase HPLC.
HPLC Analysis - An analytical RP-C18
column (Luna 18(2), 5 µm, 150 by 4.6 mm;
Phenomenex, Aschaffenburg, Germany) was
used at a flow rate of 0.6 ml min-1
on a Waters
600 HPLC-system. The mobile phase comprised
a mixture of two solvents: A, 30 % (v/v)
acetonitrile; B, 80 % (v/v) 2-propanol. The
separation was performed with a linear gradient
of solvent B from 15 to 80 % within 20 min and
additionally from 80 to 95 % within 10 min. The
detection of UV absorbance was performed
routinely at 240 nm with a Waters 996
photodiode array detector.
Solubilization and Purification of the
Enzyme - All steps for solubilization and column
chromatography were performed in an anaerobic
glove box with an FPLC System (P500 pump
system, Amersham Pharmacia Biotech) at 8 °C.
The purification of C25 dehydrogenase started
with solubilization of the membrane-bound
protein fraction. The crude extract routinely
obtained from 40 g cells (wet mass) was
centrifuged at 150,000 x g, and the membrane-
bound protein fraction in the pellet was
resuspended in two volumes of buffer containing
20 mM Tris/H3PO4 (pH 7.0). The mixture was
stirred gently, and Tween 20 (70 % w/v in H2O)
was slowly added to a final concentration of 2
mg detergent (mg membrane protein)-1
. Then
glycerol was added to a final concentration of 10
% (w/v), and the solution was gently stirred for 2
h at 8 °C. After centrifugation at 150,000 x g for
2 h, the supernatant was applied onto a DEAE-
Sepharose column.
DEAE - Sepharose fast flow column (70 ml
volume) was equilibrated with 10 column
volumes of buffer A (20 mM Tris/H3PO4 (pH
7.0), 1 mM dithiothreitol (DTT), and 0.02 %
Tween 20). The solubilized membrane protein
fraction (672 mg protein) was applied onto the
DEAE column at a flow rate of 2 ml min-1
, and
the column was washed with 5 volumes of buffer
A, then with 5 volumes of buffer B (20 mM 2-
(N-morpholino)ethanesulfonic acid (MES)/Tris
(pH 6.0), 1 mM DTT, and 0.02 % Tween 20),
and additionally with 7 volumes of buffer B
containing 50 mM KCl. The active protein pool
was eluted with 1-2 volumes of buffer B
containing 100 mM KCl. Resource Q (Amersham Pharmacia Biotech)
column (6 ml volume) was equilibrated with 10
column volumes of buffer A. The active protein
pool after chromatography on DEAE-Sepharose
column (176 mg protein) was diluted with an
equal volume of buffer A and applied onto the
Resource Q column at a flow rate of 2 ml min-1
.
The column was washed with 3 volumes of
buffer A, then with 2 volumes of buffer B, and
additionally with 5 volumes of buffer B
containing 50 mM KCl. The active protein pool
was then eluted with 6-7 volumes of buffer B
containing 75 mM KCl.
Reactive Green 19 - Agarose (Sigma) column
(30 ml volume) was equilibrated with 10 column
volumes of buffer B. The active protein pool
after chromatography on the Resource Q column
(32 mg protein) was concentrated fourfold using
centrifugal devices (Pall, MicrosepTM
, 30 kDa),
diluted with a threefold volume of buffer B, and
concentrated fourfold again to reduce the salt
concentration. Then the concentrated protein
pool was diluted tenfold with buffer B, and
applied onto the column that was run at a flow
rate of 1 ml min-1
. The column was washed with
3 volumes of buffer B, then with 3 volumes of
buffer A, and the active protein pool was eluted
with 8-10 volumes of buffer A containing 2 mM
cholic acid.
Reactive Red 120 - Agarose (Sigma) column (10
ml volume) was equilibrated with 10 column
volumes of buffer B. The active protein pool
after chromatography on Reactive Green 19
column was concentrated fourfold by centrifugal
devices (Pall, MicrosepTM
, 30 kDa), diluted with
a ninefold volume of buffer B to reduce the
concentration of cholic acid, and applied onto the
column at a flow rate of 1 ml min-1
. The column
was washed with 2 volumes of buffer B, and the
enzyme was eluted with 1 volumes of 50 % of
buffer A. The fraction was concentrated by
centrifugal devices (Pall, MicrosepTM
, 30 kDa).
Preparation of cholest-4-en-3-one-25-ol
- C25 dehydrogenase assays (0.5 ml) were
performed for the synthesis of cholest-4-en-3-
one-25-ol. The reaction mixtures were incubated
for 14 h and were then extracted with three
volumes of ethyl acetate. The ethyl acetate
fractions were evaporated, the pellet was
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resuspended in 0.2 ml of 2-propanol, and applied
to a HPLC column. The peaks containing
product were collected, and the solution was
lyophilized. The obtained cholest-4-en-3-one-25-
ol was dissolved in 0.3 ml 1,4-dioxane, and the
concentration was determined by using a
calibration curve prepared with cholest-4-en-3-
one.
Characterization of the Enzyme -
Electron acceptor specificity of the C25
dehydrogenase was tested by using the following
electron acceptors: K3[Fe(CN)6], ferricenium
tetrafluoroborate, ferricenium hexafluoro-
phosphate, NAD+, NADP
+, phenazine metho-
sulphate, 2,6-dichlorophenol-indophenol,
methylene blue and duroquinone. HPLC-based
assays were set up with 5 mM each of the
electron acceptor. To assess the pH optimum,
C25 dehydrogenase was assayed in 100 mM
potassium phosphate buffer within a pH range of
6.0 – 8.0. Reversibility of the C25 hydroxylation
reaction was tested under anaerobic conditions in
100 mM potassium phosphate buffer (pH 7.5),
containing 10 mM methyl viologen, 5 mM
dithionite and 0.1 mM cholest-4-en-3-one-25-ol.
After adding the enzyme (4.5 µg), the reaction
mixture was incubated for 16 h and then
analyzed by HPLC. UV-visible spectra were
recorded with a Cary spectrophotometer (Cary
100 Bio, Varian) by using a gas-tight stoppered
quartz cuvette under anaerobic conditions. The
native molecular mass of C25 dehydrogenase
was determined by gel filtration on a Superdex
200 column (24 ml volume, Amersham
Pharmacia Biotech) at a flow rate 0.4 ml min-1
.
The column was equilibrated with buffer
containing 20 mM Tris/H3PO4 (pH 7.0), 1 mM
DTT, 500 mM KCl, and 0,02 % Tween 20. For
calibration thyroglobulin (669 kDa), ferritin (440
kDa), aldolase (158 kDa), bovine serum albumin
(69 kDa), chymotrypsinogen A (25 kDa), and
RNaseI (13.7 kDa) were used. N-terminal
labelling of native C25 dehydrogenase was
carried out by derivatization with ((N-
succinimidyloxycarbonylmethyl)tris(2,4,6-
trimethoxyphenyl) phosphonium bromide)
(TMPP-Ac-OSu) according to (14). MASCOT
MS/MS data searches were performed by using
the protein database derived from the genome
sequences of S. denitrificans (Dermer & Fuchs,
unpublished data).
Computational Analysis - The BLASTP
searches were performed via the NCBI BLAST
server (www.ncbi.nlm.nih.gov) (15,16). The
search was performed in September 2011. The
amino acid sequences of subunits of C25
dehydrogenase were used as queries for
BLASTP searches against assembled bacterial
genomes. Gene prediction and annotation of the
draft genome was carried out using the RAST
server (17) and Geneious package v5.4 for
manual correction (18). For construction of a
phylogenetic tree, the amino acid sequences
were aligned using ClustalW implemented
within MEGA4 (19). The multiple alignments
were performed using ClustalW implicated into
BioEdit package (version 7.0.9.0).
Other methods - Protein concentrations
were determined with a BCA protein
quantification kit (VWR, Darmstadt) with bovine
serum albumin as standard. Discontinuous SDS-
PAGE was performed in 12 % (w/v) polyacryl-
amide gels according to standard procedures
(20). Blue native PAGE was performed in 8 %
(w/v) polyacrylamide gels according to modified
standard procedures (21). Cathode buffer
contained 25 mM Tris/glycine (pH 8.4) and 0.02
% or 0.002 %, respectively, of Coomassie G250.
Anode buffer contained 25 mM Tris/glycine (pH
8.4). Sample buffer contained 0.25 M Tris/Cl
(pH 6.8), 20 % glycerol and traces of
bromophenol blue. The gel was run at 8 °C and
10 mA. Gels were stained by Coomassie
Brilliant Blue R-250. Image Lab software (Bio-
Rad version 2.0 build 8) was used for analysis of
subunits composition based on their relative
abundance in SDS-gel. MS analysis of excised
gel bands was carried out as described (22).
Photometric determinations of iron and inorganic
sulphide were performed by standard chemical
techniques (23). Additionally, a simultaneous
determination of 32 elements in the purified
enzyme was performed by inductively coupled
plasma optical emission spectroscopy (ICP-OES)
using a Jarrel Ash Plasma Comp 750 instrument
at the center of Complex Carbohydrate Research,
University of Georgia, USA. The identification
of the nucleotide moiety of the molybdenum
cofactor was performed by using a Lichrospher
100 RP-18 E column (5 μm particle size, 4 × 125
mm) as described (24).
Sequences - The sequence data of new
identified molybdoenzymes reported in this work
have been submitted to GenBank and have been
assigned the accession numbers: JQ292991
(S25dA); JQ292992 (S25dB); JQ292993
(S25dC); JQ292994 (S25dA2); JQ292995
(S25dA3); JQ292996 (S25dA4); JQ292997
(S25dB4); JQ292998 (S25dC4); JQ292999
(S25dD4); JQ293000 (S25dA5); JQ293001
(S25dB5); JQ293002 (S25dC5); JQ293003
(S25dA6); JQ293004 (S25dB6); JQ293005
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(S25dC6); JQ293006 (S25dA7); JQ293007
(S25dB7); JQ293008 (S25dC7); JQ293009
(EbdA-like); JQ293010 (EbdB-like); JQ293011
(EbdC-like); JQ293012 (EbdD-like).
RESULTS
C25 dehydrogenase (hydroxylase)
activity in Sterolibacterium denitrificans - An
HPLC-based assay with cholest-4-en-3-one as
substrate and potassium hexacyanoferrate (III) as
artificial electron acceptor was developed for
activity measurements in cell extracts and during
purification of the enzyme. Transformation of
the substrate to products was monitored routinely
at 240 nm (SI Fig. 1). With cell extract, 25-
hydroxy-cholest-4-en-3-one was formed as
product, but in addition cholesta-1,4-dien-3-one
and 25-hydroxy-cholesta-1,4-dien-3-one were
formed (SI Fig. 1B), which is due to the presence
of the initial enzymes of the pathway (see Fig.
1). These initial enzymes act also on compounds
I and II (Fig. 1). In contrast, purified C25
dehydrogenase produced only 25-hydroxy-
cholest-4-en-3-one (SI Fig. 1C, see below).
Coupling of substrate oxidation to the reduction
of the artificial electron acceptors 2,6-
dichlorophenol-indophenol or phenazine
methosulfate in a spectrophotometric test could
not be used with cell extracts due to unspecific
reactions occurring in extracts. A photometrical
assay with ferricenium cation as artificial
electron acceptor, however, was developed to
measure the activity of purified C25
dehydrogenase (see below). Activity was linearly
dependent on the amount of added protein in the
range 0 – 0.11 mg protein ml-1
and required the
presence of detergents like Triton X100, Tween
20, dodecyl β-D-maltoside, or 2-hydroxypropyl-
β-cyclodextrin (briefly cyclodextrin). Routinely,
20 % (w/v) cyclodextrin was added to dissolve
up to 0.7 mM of steroid substrates, and addition
of detergent or cyclodextrin resulted in a
thousand-fold activity increase. The pH optimum
of the reaction was between 7.0 and 7.5. Nearly
identical activities were obtained under oxic and
strictly anoxic conditions, indicating that
molecular oxygen is not required for the
hydroxylation of the side chain. The specific
hydroxylation rate in cell extract was 2 nmol
min-1
(mU) (mg protein)-1
. This value is close to
the calculated cholesterol degradation rate of 3
mU (mg protein)-1
, when cells were growing
under denitrifying conditions with a doubling
time of 44 h. Similar activities were measured in
cells that were grown on cholesterol under oxic
or anoxic (denitrifying) conditions. This
indicates that S. denitrificans may use the same
anaerobic degradation strategy both for
anaerobic and aerobic growth on cholesterol.
This conclusion is corroborated by genome
sequencing (see below).
Subcellular localization - When freshly
harvested cells were treated with polymyxin B
and lysozyme in hypotonic medium to gently
lyse the cells, formation of cell ghosts was
observed by microscopic examination. After
centrifugation at 150,000 x g, 60 % of C25
dehydrogenase activity and 83 % of cholest-4-
en-3-one-∆1-dehydrogenase activity were found
in the membrane fraction, whereas 90 % of
malate dehydrogenase activity was recovered in
the soluble protein fraction. Furthermore, after
centrifugation of cell extract on a sucrose
gradient, C25 dehydrogenase activity was
completely recovered in the cytoplasmic
membrane fraction, whereas 85 % of the
cytoplasmic marker protein malate
dehydrogenase was recovered in the soluble
protein fraction. To address, whether the electron
acceptor site of the enzyme faces the periplasmic
or cytoplasmic side, whole cell assays were
carried out. Note that the cytoplasmic membrane
is not permeable for charged ions like the
electron acceptors potassium hexacyanoferrate or
NAD+. Nevertheless, similar C25 dehydrogenase
activities were measured with whole cells and
with cell extract, whereas only residual activity
of the cytoplasmic marker enzyme malate
dehydrogenase (NAD+ dependent) was observed
when tested with whole cells (Fig. 2). Addition
of high concentrations (1 M) of the non-
chaotropic salt NaCl to the membrane fraction
did not solubilize the enzyme, whereas after
treatment with Tween 20, the C25 hydroxylation
activity was observed solely in the solubilized
protein fraction. These observations indicate that
C25 dehydrogenase is preferentially associated
with the cytoplasmic membrane with the electron
accepting site facing the periplasmic space.
Purification - The membrane-bound
protein fraction was used for purification
yielding a brownish enzyme preparation.
Routinely, the soluble protein fraction after
Tween 20 treatment was not used further; it
contained approximately 40 % of total C25
dehydrogenase activity and can be used for
enzyme purification following the same scheme.
The solubilization of membrane-bound protein
was performed with the weak detergent Tween
20, which is commonly used for solubilization of
peripheral membrane proteins. After
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solubilization, about 95 % of the activity was
obtained in the solubilized protein fraction,
whereas only residual activity could still be
observed in the membrane fraction. A nearly
homogeneous C25 dehydrogenase fraction
containing three subunits was obtained after four
chromatographic steps (Fig. 3A, SI Table 1).
Two chromatographic steps were carried out on
DEAE and Resource Q anion exchange columns,
followed by two affinity chromatographic steps
using Reactive Green 19 and Reactive Red 120.
C25 dehydrogenase was eluted from Reactive
Green 19 by the substrate analogue cholic acid,
whereas a pH-shift was used for its elution from
Reactive Red 120. Although the enzyme activity
was not affected by oxygen in cell extracts, after
the first chromatographic DEAE step the
enriched fraction quickly lost activity in air.
Therefore purification had to be carried out
under anoxic conditions.
Molecular properties - Steroid C25
dehydrogenase consists of three subunits of
approximately 108 kDa, 38 kDa, and 27 kDa, as
revealed by SDS-PAGE (Fig. 3A). The subunits
were present in an approximate 1:1:1 molar ratio.
The molecular mass of the native enzyme was
determined by gel filtration as 168 ± 12 kDa
indicating a αβγ-composition. Based on peptide
mass fingerprint analysis of gel bands the
corresponding genes were identified in a draft
genome of the organism (Dermer & Fuchs,
unpublished data) (Fig. 3B). The gene for the
alpha subunit was a single ORF related to the
large subunit of ethylbenzene dehydrogenase,
which contained a 5' sequence coding for an N-
terminal twin-arginine translocation (Tat) leader
peptide. In contrast, the genes for the beta and
gamma subunits were located 396 kbp away in a
cluster of three genes coding for α, β, and γ
subunits of another molybdenum-containing
enzyme also related to ethylbenzene
dehydrogenase. Apparently, the beta and gamma
subunits are shared by these two similar
molybdenum enzymes, one being C25
dehydrogenase, the function of the other being
unknown. The phenomenon of subunit sharing
may be common in this metabolic pathway (see
Discussion). The calculated sizes of the subunits
were 108 kDa (alpha), 38 kDa (beta), and 23
kDa (gamma). The reason for the apparent larger
size of the gamma subunit estimated by SDS-
PAGE may be due to hydrophobicity of this
putative membrane anchor protein.
Edman degradation for N-terminal
amino acid sequencing of the proteins after
blotting on a PVDF-membrane or electroelution
out of SDS-PAGE gels was not successful. This
failure could be caused by blocking of the α-
amino group. The N-terminal amino acid of the
alpha subunit could, however, be derivatized
with TMPP-Ac-OSu. After mass spectrometric
detection of labeled peptides, the TMPP-label
was identified on the methionine of the large
subunit corresponding to the first amino acid
coded by the gene. Surprisingly, in this enzyme
preparation the N-terminal leader peptide of the
α subunit apparently was not cleaved off. No
TMPP-label could be identified on the beta and
gamma subunits indicating that they were
probably not accessible for derivatization.
The molar contents of molybdenum,
iron, and acid-labile sulfur were determined. The
molybdenum content was 0.7 ± 0.1 mol (mol of
enzyme)-1
as determined by inductively coupled
plasma optical emission spectrometry (ICP-
OES), the iron content was 16 ± 5 mol (mol of
enzyme)-1
as determined by ICP-OES and
colorimetric assay, and the acid-labile sulfur
content was 18 ± 5 mol (mol of enzyme)-1
. These
values are consistent with the predicted presence
of 1 molybdenum atom, 4 [Fe4S4], and one
[Fe3S4]-clusters, and 1 heme. Analysis of the
nucleotide part of molybdenum cofactor revealed
the presence of GMP rather than AMP or CMP.
Spectral properties - The complex UV-
visible spectrum of the purified brownish
enzyme showed distinct absorption maxima at
424, 527, and 561 nm, which indicated the
presence of a reduced heme b cofactor, as well as
a broad shoulder around 400 nm. Anaerobic
oxidation of the enzyme with potassium
hexacyanoferrate (III) resulted in the
disappearance of α and β peaks of the heme at
561 and 527 nm and the shift of the Soret band at
424 nm to 416 nm (Fig. 4A). The difference
spectrum of reduced-minus-oxidized enzyme
indicated the presence of a heme b cofactor (Fig.
4B). The spectrum of the reduced enzyme could
be restored by addition of the substrate cholest-
4-en-3-one to the cyanoferrate-oxidized enzyme
(Fig. 4A). The substrate-reduced enzyme and the
enzyme as isolated showed identical heme b
spectra, suggesting that C25 dehydrogenase was
purified in the reduced heme form. Further
treatment of the enzyme with dithionite did not
result in further reduction of the heme cofactor,
but resulted in further bleaching of the
absorption between 400 and 500 nm (Fig. 4A),
which is indicative for the presence of iron-
sulfur clusters. Obviously, these clusters are not
fully reduced by the substrate and need a strong
chemical reductant such as dithionite for being
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entirely reduced. The heme content of the
enzyme was determined as 1.13 mol (mol
protein)-1
from dithionite-reduced enzyme, using
a molar absorption coefficient ε556 nm of 34,700
M-1
cm-1
for the α band (25).
Catalytic properties - The purified en-
zyme catalyzed the hydroxylation of the tertiary
C25 atom of the steroid side chain. The low
water solubility of the substrates, which required
the addition of detergents or cyclodextrin,
hampered the determination of kinetic properties
of C25 dehydrogenase. An HPLC based assay or
a photometrical assay containing ferricenium
cation as artificial electron acceptor was used to
measure activity of the purified enzyme in the
presence of cyclodextrin. The substrate routinely
used was cholest-4-en-3-one (SI Fig. 1A). Only
25-hydroxy-cholest-4-en-3-one was formed (SI
Fig. 1C). The stoichiometry was 1 mol C25
hydroxylated product formed per 1.8 mol
ferricenium added. Addition of water soluble
organic solvents like 1,4-dioxane or
dimethylsulfoxide to the reaction mixture to
increase the solubility of the substrates resulted
in strong decrease of the enzymatic activity.
Addition of detergents or cyclodextrin to the
assay resulted in thousand-fold increased
activity.
A specific activity of 220 nmol min-1
mg-
1 was found with cholest-4-en-3-one as substrate
at an optimal pH value of 7-7.5 and in the
presence of cyclodextrin; this activity is in the
same range as that reported for ethylbenzene
dehydrogenase (12). A slightly higher
hydroxylation rate was observed with cholesta-
4,6-dien-3-one, whereas lower activity was
observed with cholesterol, cholest-5-en-3β-ol-7-
one, and cholcalciferol (Table 1). No activity
could be measured with ergosterol, desmosterol,
or cholesterylbenzoate; the enzyme also did not
act on isoamylbenzoate, isoamyl alcohol or 2-
methyl-butane that mimic the branched side
chain of the steroids. The potential of C25
dehydrogenase to catalyze the reverse reaction
was tested by an anaerobic enzyme assay with
reduced methyl viologen as electron donor and
cholest-4-en-3-one-25-ol as substrate; no
reduction to cholest-4-en-3-one was detected.
The enzyme was not inhibited by sodium azide
or sodium cyanide (5 mM each).
The capability of bovine heart
cytochrome c to serve as an electron acceptor
was tested in a modified spectrophotometrical
assay. Cytochrome c was indeed reduced, the
specific enzyme activity being 20 nmol min-1
mg-1
with cholest-4-en-3-one as a substrate
which is 10-fold lower than with
hexacyanoferrate (III). The Km value for bovine
heart cytochrome c was estimated as 15 µM. Stability - The activity of C25 dehydro-
genase was not affected by aerobic preparation
of cell extracts, whereas the purified enzyme was
inactivated irreversibly by incubation in air with
a half-life of 14 min (SI Fig. 2). The inactivation
was largely prevented by addition of the artificial
electron acceptor potassium hexacyanoferrate
(III) to the enzyme preparation. In presence of
cyanoferrate, more than 90 % of the enzyme
activity was retained after two hours incubation
in air; still, the enzymatic activity was
completely lost after 24 h of incubation. The
enzyme can be stored for at least 3 months at 4
°C under anaerobic conditions.
Sequence analysis and phylogeny -
Analysis of the α subunit of C25 dehydrogenase
showed high similarity to the α subunit of
ethylbenzene dehydrogenase of Aromatoleum
aromaticum (35 % amino acid sequence identity,
accession number CAI07432), an unidentified
molybdopterin oxidoreductase of Desulfococcus
oleovorans strain Hxd3 (35 % amino acid
sequence identity, accession number
ABW66004), and further molybdopterin
containing oxidoreductases of type II group of
the DMSO reductase family. Examination of the
N-terminus of the α subunit revealed that it
contained a ‘twin arginine’ motif,
MQISRRQFIV (Fig. 5), indicative of proteins
that bind a prosthetic group and fold in the
cytoplasm, before the translocation via the Tat
system (26) . Furthermore a cysteine-rich motif,
GTHTRANCIGACSWDV-(26)-NPRGCQK,
was identified in the N-terminal part of the large
subunit (Fig. 5). This motif is characteristic of
type II enzymes with an aspartate molybdenum
ligand in the active site and is responsible for
coordination of a [Fe4S4]-cluster (27) . The β
subunit showed high similarity to the β subunit
of ethylbenzene dehydrogenase (55 % amino
acid sequence identity) and to further iron-sulfur
subunits of DMSO reductase like proteins.
Sequence analysis revealed conserved cysteines
(Fig. 6; SI Fig. 4), which are responsible for
coordination of four [Fe-S]-clusters (28).
Analysis of amino acid sequence of the γ subunit
showed similarity exclusively to the γ subunit of
ethylbenzene dehydrogenase and ethylbenzene
dehydrogenase-like proteins. Highly conserved
methionine (M111) and lysine (K203) residues
were identified (SI Fig. 5) which are the axial
ligands of the heme b iron in ethylbenzene
dehydrogenase (29). Furthermore, neither azide
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nor cyanide inhibit the C25-Hydroxylase. This
finding supports the assumption that the heme is
hexacoordinated.
Genes coding for steroid C25
dehydrogenase-like enzymes in the genome - S.
denitrificans harbors a set of genes coding for at
least seven further C25 dehydrogenase-like
enzymes (Fig. 7), which are scattered in the
genome. Genes of six C25 dehydrogenase-like
enzymes are organized in gene clusters coding
for α, β, and γ subunits, whereas an additional
gene for a single α subunit is located as a single
ORF, like the single gene for the alpha subunit of
steroid C25 dehydrogenase (Fig. 7).
Interestingly, only two genes encoding
maturation chaperones were identified in the
draft genome. Maturation of complex
molybdoenzymes requires enzyme-specific
chaperones for correct insertion of co-factors
before folding and translocation across the
membrane. The private chaperone genes often
occur in the gene clusters of respective
molybdoenzyme, as it was shown for all type II
enzymes (30-33). In contrast, the two maturation
chaperones identified in the genome of S.
denitrificans may be responsible for the
maturation of eight molybdoenzymes.
Interestingly, no gene resembling any
oxygenase gene from the known aerobic steroid
metabolism could be found. This is another
argument for the functioning of the O2
independent steroid pathway studied here, both
under anoxic and oxic conditions.
DISCUSSION
Function of C25 dehydrogenase -
Previous studies showed that under anoxic
conditions cholesterol is transformed to cholest-
4-en-3-one and cholesta-1,4-dien-3-one (Fig. 1)
(11). These transformations are similar to those
occurring in aerobic steroid degradation (5).
Here we described a novel molybdenum-
containing enzyme that catalyzes the subsequent
anaerobic hydroxylation of the C25 tertiary
carbon atom of the steroid side chain. It acts as
counterpart to C26 monooxygenase of aerobic
catabolism (34) that exploits the ability of
activated molecular oxygen to overcome the high
C-H bond stability. C25 dehydrogenase
resembles ethylbenzene dehydrogenase that
catalyzes a similar oxygen-independent
hydroxylation of a hydrocarbon, forming
secondary alcohols of a wide range of aromatic
and heterocyclic compounds with an ethyl or
propyl moiety (12). Hydroxylation may proceed
via formation of a carbocation, which is
stabilized by an adjacent aromatic ring (35).
Because of the high similarity of C25
dehydrogenase with ethylbenzene
dehydrogenase, we assume that C25
dehydrogenase employs a similar hydroxylation
mechanism. Owing to the higher stability of the
C25 carbocation compared to the terminal C26
or C27, its hydroxylation is probably favored.
S. denitrificans is a facultative anaerobic
cholesterol degrader. It appears that the same
anaerobic strategy is used under anoxic and oxic
conditions as indicated by the following
findings. (i) Comparable anaerobic C25
hydroxylation activities were measured in
extracts of anaerobically and aerobically grown
cells. (ii) Previous proteome analyses revealed
no differences in soluble protein patterns of
anaerobically and aerobically grown cells (9).
(iii) No oxygenase genes known from aerobic
cholesterol metabolism could be identified in the
draft genome of the organism. The usage of one
common anaerobic strategy is highly economic
for an organism growing on steroids both under
anoxic and oxic conditions. It reflects an
adaptation of a facultative anaerobic organism to
frequent periodical oxygen fluctuations allowing
the instantaneous usage of cholesterol, no matter
whether oxygen is available or not. Apparently,
the enzymes of the anaerobic strategy are
sufficiently oxygen-stabile in vivo and possibly
also in cell extracts, even though purified C25
dehydrogenase is oxygen labile.
Membrane association and catalysis at
lipid-water interface - C25 dehydrogenase
operates on highly hydrophobic substrates
which, owing to their low water solubility, likely
concentrate in the membrane bilayer rather than
in the aqueous phase of the cytoplasm. Thus, a
membrane associated C25 dehydrogenase can
easily reach the hydrophobic side chain of its
substrate (36), which may provide kinetic
advantages since both enzyme and its substrate
are concentrated within the membrane. A similar
situation was described for cytochrome P-450scc,
a mono-oxygenase cleaving the side chain of
cholesterol and forming pregnenolone. C25
dehydrogenase may partially immerse into the
membrane bilayer thus facilitating the binding of
cholesterol (37).
C25 dehydrogenase operates at the lipid-
water interface on lipophilic substrates; sequence
analysis does not predict transmembrane helices.
All attempts to establish an in vitro assay failed
when cholest-4-en-3-one was simply added to
the assay. However, the activity increased a
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thousand-fold when detergents or cyclodextrin
were added to the reaction mixture. The ability
of detergents to increase activity is well-known
for enzymes operating on hydrophobic
substrates. The phenomenon of interfacial
activation was described for lipases (38),
whereas the detergent dependent activation of
tyrosinase (39) or pyruvate oxidase (40) is
thought to be caused by protein-detergent
interaction. We suggest that the activation effect
on C25 dehydrogenase is caused by increased
substrate solubility rather than protein-detergent
interaction, as a comparable stimulation of C25
hydroxylation activity was achieved both with
detergents and cyclodextrin.
Electron acceptor - The estimated redox
potential of the C25-hydroxycholesterol/chole-
sterol pair is around + 30 mV, based on
theoretical calculations of related
alcohol/hydrocarbon couples. Steroid C25
dehydrogenase shows in vitro activity only with
artificial electron acceptors of high redox
potential, like cyanoferrate (E°’ = + 420 mV) or
ferricenium ion (E°’ = + 380 mV). The
difference in redox potential of the
substrate/product pair and that of the electron
acceptor may explain the irreversibility of the
reaction under experimental conditions. Due to
the accessibility of the electron acceptor site of
C25 dehydrogenase from the periplasmic space,
a periplasmic c type cytochrome of similar
positive redox potential may function as natural
electron acceptor, coupling C25 hydroxylation to
nitrate reduction or alternatively to O2 reduction.
The use of cytochrome c was also proposed for
the closely related ethylbenzene dehydrogenase
from A. aromaticum (12). Bovine heart
cytochrome c, tested here, is probably a poor
surrogate for the bacterial cytochrome c.
Substrate specifity - Steroid C25 dehydro-
genase catalyzes the hydroxylation of cholesterol
but also of intermediates II and III (Fig. 1).
Apparently, the first steps of anaerobic
degradation of cholesterol proceed randomly via
independent reaction steps operating in parallel
on the side chain and the ring system. A similar
situation is known for aerobic steroid
degradation (5). Thus, it appears to be a general
strategy of microbial steroid decomposition to
follow a branched route of reactions. The
hydroxylation rate of cholesterol was lower
compared to that of cholest-4-en-3-one or
cholesta-4,6-dien-3-one (Table 1), all substrates
having the same side chains but differing in the
ring systems (SI Fig. 3). Presence and
localization of double bonds have an impact on
the conformation of the molecule. Thus, the
more planar conformations of rings A and B of
cholest-4-en-3-one and cholesta-4,6-dien-3-one
may result in a better fitting into the active site of
the enzyme. No hydroxylation activity was
observed with stigmasterol and ergosterol, whose
side chains structures differ from that of
cholesterol (SI Fig. 3). They harbor additional
methyl or ethyl substituents at C24 which may
cause sterical hindrances in the active site.
Therefore, we assume that C25 dehydrogenase
operates selectively on the isooctane side chain
of steroids and requires a tertiary C25.
One may ask whether there is a
physiological reason for the preference of
cholest-4-en-3-one as substrate. It is well known
that cholesterol is an important constituent of
eukaryotic membranes. However, steroids can
affect the growth of some microorganisms,
damaging their cytoplasmic membranes, e. g. the
presence of cholest-4-en-3-one is toxic for
Mycobacterium tuberculosis (41). Therefore, a
higher C25 transformation rate of 3-ketosteroids
may reflect an adaptation of S. denitrificans to
remove toxic intermediates from the membrane.
Hydroxylation at C25 increases the water
solubility of the molecules, thus facilitating
migration out of the membrane (42).
C25 dehydrogenase, a new member of
the DMSO reductase family of molybdoenzymes
- Steroid C25 dehydrogenase is a molyb-
denum/iron-sulfur/heme containing enzyme and
belongs to the type II of DMSO reductase family
enzymes. Based on its high sequence similarity
to ethylbenzene dehydrogenase (29) and other
archetypal complex iron-sulfur-molybdo-
enzymes (28), the α subunit contains molyb-
denum coordinated by molybdo-bis(pyranopterin
guanine dinucleotide) cofactor and the FS0-
[Fe4S4]-cluster (Fig. 5B); the β subunit harbors
FSI - FSIII-[Fe4S4], and FSIV-[Fe3S4]-clusters
(Fig. 6), and the γ subunit contains the heme b
group with conserved methionine and lysine
axial ligands. Remarkably, the genome of S.
denitrificans harbors genes coding for at least
seven further C25 dehydrogenase-like enzymes
(Fig. 7). The large subunits of all of them
possess a signal peptide for Tat-dependent
translocation (Fig. 5A) and therefore may be
located in the periplasmic space. A phylogenetic
tree (Fig. 8) reveals that steroid C25
dehydrogenase and related enzymes form a
distinct clade, together with other heterotrimeric,
periplasmic enzymes like ethylbenzene dehydro-
genase, selenate reductase, dimethylsulfide
dehydrogenase, and chlorate reductase.
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A signature of all type II enzymes is the
presence of the FS0-[Fe4S4]-cluster in the
catalytic subunit, which is coordinated by an N-
terminal consensus sequence (CA/HA)-x2-3-CB-x3-
CC-x27-34-CD-x-K/R (27). So far, ethylbenzene
dehydrogenase represents an exception with a
HA-x3-CB-x5-CC-x34-CD-x-K consensus sequence.
Interestingly, the consensus sequence of C25
dehydrogenase and related enzymes of S.
denitrificans also differs from that of the other
type II enzymes (Fig. 5B). Ethylbenzene
dehydrogenase and C25 dehydrogenase act on
hydrophobic substrates, in contrast to other type
II enzymes that are involved in anaerobic
respiration. The identified differences in
consensus sequence may represent a signature of
the new clade of type II enzymes.
Molybdenum hydroxylases as
counterparts of oxygenases - Degradation of
cholesterol is challenging due to its complex
structure and requires, therefore, several
activation steps. Under aerobic conditions this
part is taken over by four oxygenases initiating
the degradation of the side chain and the
cleavage of the ring structures (5). In S.
denitrificans growing on cholesterol both under
anaerobic and aerobic conditions, molybdenum
hydroxylases obviously operate as oxygenase
counterparts. The presence of a set of eight C25
dehydrogenase-like enzymes supports this
suggestion. These paralogous enzymes may have
evolved by several gene duplication events.
Molybdenum hydroxylases probably represent a
general strategy of facultative microorganisms to
activate hydrophobic substrates containing a
sterane ring.
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36. Rothman, J. E., and Engelman, D. M. (1972) Nat. New Biol. 237, 42-44
37. Seybert, D. W., Lancaster, J. R., Lambeth, J. D., and Kamin, H. (1979) J. Biol. Chem. 254,
2088-2098
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FOOTNOTES
* This work was supported by Deutsche Forschungsgemeinschaft. We thank Michael Müller and
Volker Brecht, Freiburg, for mass spectrometry measurements, and Yin-Ru Chiang and Wael Ismail,
Freiburg, for their invaluable contributions during the early stages of this work.
FIGURE LEGENDS
Fig. 1. Initial steps of anaerobic cholesterol metabolism (acm) in S. denitrificans. AcmA -
cholesterol dehydrogenase/isomerase; AcmB - cholest-4-en-3-one-∆1-dehydrogenase; S25DH - steroid
C25 dehydrogenase. (I) - cholesterol, (II) - cholest-4-en-3-one, (III) - cholesta-1,4-dien-3-one, (IV) -
25-hydroxy-cholesterol, (V) - 25-hydroxy-cholest-4-en-3-one, (VI) - 25-hydroxy-cholesta-1,4-dien-3-
one. Conversion of intermediate (IV) to intermediate (V) was not experimentally proven (dashed
arrow).
Fig. 2. Cellular localization of C25 dehydrogenase. (A) C25 dehydrogenase assay with cell
suspension and cell extract. The assay mixture contained 0.1 M potassium phosphate buffer (pH 7.5),
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2 M sucrose, 7 % (w/v) (2-hydroxypropyl)-β-cyclodextrin, 0.7 mM cholest-4-en-3-one, 5 mM
K3[Fe(CN)6], and 0.1 ml cell suspension (0.3 mg protein) or cell extract (0.3 mg protein) in a total
volume of 0.3 ml. Solid line - assay with cell suspension, dashed line - assay with cell extract. (x) -
conversion of cholest-4-en-3-one; (●;○) - formation of cholesta-1,4-dien-3-one; (■;□) - formation of
25-hydroxy-cholesta-1,4-dien-3-one. (B) Relative activities of enzymes determined in cell suspension
and in cell extract. Solid bars - activities in cell extract: C25 dehydrogenase (2.2 mU mg-1
), cholest-4-
en-3-one-∆1-dehydrogenase (6 mU mg
-1), and malate dehydrogenase (1 U mg
-1); open bars - activities
measured with suspension of whole cells: C25 dehydrogenase (1.5 mU mg-1
), cholest-4-en-3-one-∆1-
dehydrogenase (6 mU mg-1
), and malate dehydrogenase (72 mU mg-1
).
Fig. 3. Purification and characterization of C25 dehydrogenase.
A. SDS-PAGE (11 %) of active pools during purification of C25 dehydrogenase (I) and blue
native-PAGE of the purified enzyme (II). I. Lane 1, cell extract (100 µg of protein); Lane 2,
solubilized membrane fraction (100 µg of protein); Lanes 3-5, active pools after chromatography on
DEAE (L3, 34 µg of protein); on Resource Q (L4, 20 µg of protein); on Reactive Red 120 (L5, 6 µg of
protein); Lane M, marker proteins (sizes given in right margin).
II. (A) Blue native-PAGE (8 %) of enzyme after Reactive Red 120 chromatography (9 µg). (B) The
visible band was cut out, treated with SDS-buffer, and analyzed by SDS-PAGE (11 %). Proteins were
stained with sensitive Coomassie blue.
B. Organization of genes encoding C25 dehydrogenase and C25 dehydrogenase-like enzyme in S.
denitrificans. (S25dA) alpha subunit of C25 dehydrogenase; (S25dB) beta subunit of C25
dehydrogenase; (S25dC) gamma subunit of C25 dehydrogenase; (S25dA3) molybdenum-containing
subunit of a C25 dehydrogenase-like protein of unknown function.
Fig. 4. UV-visible spectrum of C25 dehydrogenase. [A], spectrum of purified enzyme (0.9 mg ml
-
1). (a) Directly after purification under anaerobic conditions, (b) after anaerobic oxidation with 170
µM potassium hexacyanoferrate, (c) after re-reduction with 50 µM cholest-4-en-3-one dissolved in
dioxane, and (d) after vigorous reduction with dithionite. For better visibility, spectra b-d were offset
along the y axis (+ 0.01, + 0.02, + 0.03, respectively). [B], difference spectrum of the reduced
enzyme (a in panel A) minus potassium hexacyanoferrate-oxidized enzyme (b in A).
Fig. 5. Sequence alignment of the molybdenum-containing subunit of C25 dehydrogenase from
S. denitrificans, of ethylbenzene dehydrogenase from A. aromaticum, and of related enzymes.
(A) Alignment of the N-terminal sequences bearing a consensus motif for the Tat export way. The
conserved amino acids are highlighted.
(B) Alignment of the sequence surrounding the [Fe4S4]-cluster, which is characteristic of type II
enzymes of the DMSO reductase family. Highly conserved amino acids coordinating this FS0-[Fe4S4]-
cluster are highlighted. S25dA: alpha subunit of C25 dehydrogenase; EbdA: alpha subunit of
ethylbenzene dehydrogenase; S25dA2-S25dA7: molybdenum containing subunits of enzymes with
high similarity to C25 dehydrogenase, which were identified in the genome of S. denitrificans.
Fig. 6. Analysis of amino acid sequence of beta subunit of C25 dehydrogenase.
Coordination of the FS1-FS4 [Fe-S]-clusters in the beta subunit of C25 dehydrogenase, as proposed
based on the sequence similarity to ethylbenzene dehydrogenase beta subunit (EbdB) and the crystal
structure of ethylbenzene dehydrogenase.
Fig. 7. Genes coding for C25 dehydrogenase and C25 dehydrogenase-like enzymes in the genome
of S. denitrificans. Amino acid sequences of α, β, and γ subunits of C25 dehydrogenase were used as
queries for BLASTP searches against the genome of S. denitrificans. The BLASTP (2.2.14) search
tool implemented into RAST server was used. (S25dA) - molybdenum containing α subunit of steroid
C25 dehydrogenase; (S25dB) - [Fe-S]-clusters containing β subunit of steroid C25 dehydrogenase;
(S25dC) - heme b containing γ subunit of steroid C25 dehydrogenase; (α2-α7) - genes coding for
molybdenum containing subunits related to S25dA; (β4-β7) - genes coding for [Fe-S]-clusters
containing subunits related to S25dB; (γ4-γ7) - genes coding for heme b containing subunits related to
S25dB; (δ4, EbdD-like) - genes coding for proteins related to maturation chaperone of ethylbenzene
dehydrogenase. (EbdABC-like) - molybdenum containing enzyme related to S25dABC, but
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phylogenetically clustering with ethylbenzene dehydrogenase (Fig. 8). The sequence identities to the
corresponding subunits of C25 dehydrogenase are given in brackets.
Fig. 8. Phylogenetic tree of enzymes of the DMSO reductase family (type I, II, and III) based on
amino acid sequences of molybdenum-containing alpha subunits. C25 dehydrogenase and several
related enzymes in S. denitrificans belong to family II, as do ethylbenzene dehydrogenase from A.
aromaticum and related dehydrogenases. The tree was constructed using the neighbor-joining
algorithm. Bootstrap values higher than 75 % are marked with dots. The scale bar represents 0.1
changes per amino acid. GenBank accession numbers for sequences used to construct the tree are
listed in SI Table 2. Abbreviations for molybdenum-containing subunits of proteins: BisC, biotin
sulfoxide reductase; ClrA, chlorate reductase; DdhA, dimethylsulfide dehydrogenase;
DmsA/DorA/DsrA, dimethylsufoxide reductase; EbdA, ethylbenzene dehydrogenase; FdhG, formate
dehydrogenase; NarG, respiratory nitrate reductase; NapA, periplasmic nitrate reductase; NasA,
assimilatory nitrate reductase; PcrA, perchlorate reductase; PhsA, thiosulfate reductase; PsrA,
polysulfide reductase; SerA, selenate reductase; TorA, trimethylamine-N-oxide reductase.
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Table. 1: Relative activity of C25 dehydrogenase with steroid substrates. For structures of
cholesterol, cholest-4-en-3-one and cholesta-1,4-dien-3-one, see Fig. 1. Measurements of relative
activities were carried out in spectrophotometric assays. For steroid substrates also HPLC assays were
performed. No activity was observed with isoamylbenzoate, isoamylalcohol, 2-methyl-butane, ergosterol,
desmosterol, and stigmasterol. For MS-analysis the assay mixture (0.5 ml) was extracted with three
volumes of ethyl acetate. Ethyl acetate was evaporated, and the dry residue was dissolved in 100 µl of
chloroform. Electrospray ionization mass spectra (ESI-MS) were recorded with an Applied Biosystems
API 2000 triple quadrupole instrument running in positive ion mode.
n. d. - not determined
* - analysis with HPLC-ESI-MS
§ - analysis with HPLC-ESI-MS with atmospheric pressure photo ionization (APPI) source
# - formation of the product was observed using HPLC assay when the reaction mixture was incubated
for 12 h.
Substrate m/z
relative
activity
(%)
product m/z
cholest-4-en-3-one 385* (m+H) 100 401
* (m+H)
cholesta-4,6-dien-3-one 383* (m+H) 127 399
* (m+H)
5-cholesten-3β-ol-7-one 401* (m+H) 15 417
* (m+H)
cholesterol 369§ (m+H - H2O) 10 367
§ (m+H - 2 H2O)
cholecalciferol n. d. < 5# n. d.
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Fig. 1
OH
OH
OH
O
OH
O
O
OH
O
S25DH
S25DH
S25DH
AcmA
AcmB AcmB
AcmA
I
III
IV
V
VI
II
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Fig. 2
A
min
nm
ol
0
20
40
60
80
100
120
140
rela
tive
ac
tivit
y, %
B
50
100
150
200
250
300
350
0 50 100 150 200
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Fig. 3
A
B
201487 598131
( 396 kbp )S25dC S25dB S25dA3 S25dA
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Fig. 4
Data 1
400 450 500 550 6000.00
0.05
0.10
0.15
0.20
0.25
0.30
FeCN
Enzyme
S2
Dithionit
nm
Ab
s
Wavelength [nm]
Ab
so
rba
nce
∆A
A B
400 450 500 550 600
-0.020
0.005
0.030
0.055
424
561
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Fig. 5
10 20 30 40 50 60 70 80 90. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
C25dhA S.denitrificans - - - - - - - - - - - - - - - - - M Q I S R R Q F I V G S A V A A A G L G L Y S L R P K H Y V P A A P R P A D K - - - - - - - - - - - P G I P A K K V K Y N D Y S D I WR E KWKW
peg.1060 S.denitrificans - - - - - - - - - - - - - - - - - M QV S R R H F I V G T A A V A A GA G L Y S L R P K L T G P K - P G I T M P - - - - - - - - - - - P L V P A K K V K Y N D Y S D I WR E KWKW
peg.62 S.denitrificans - - - - - - - - - - M E S K P GM I GM D R R S F L K - A G GS A L A L S L C H L E L L P M N AM A Q A A GK D G N G N K A A A E L S P L E A A A E L E Y R S F E D L Y R K KWHW
peg.1309 S.denitrificans - - - - - - - - - - - - - - - - - M QV S R R N F I V G S A V A A A G L G L Y S L K P K T P A A I K A GP A L P - - - - - - - - - - - P L V S A K K I R Y N D Y S D I WR E KWKW
peg.1642 S.denitrificans - - - - - - - - M G I L A T S N L V S A S R R K F L V M A G - - - M A S A A GA A V G L F GC S R A P L Q H F K G T - - - T A S G R F D L GP R T T P K L GNWQ D L Y R Q RW T W
peg.481 S.denitrificans - - - - - - - - - M T T A S P A QP N P A R R R F L I L A G K T T V A G I A A A A T G L P GC N RM P L Q H F H G - - - - T V D G R F D L GP R T T P K L N NWQ D L Y R Q RW T W
peg.761 S.denitrificans - - - - - - - - - - - - - - M Q F M Q L T R R H F I M G S A A T V A G L A L Y S L R P R H Y V P A A P R P A D P - - - - - - - - - - - P T V P A K K V K Y N D Y S D I WR E KWKW
peg.1646 S.denitrificans - - - - - - M E R S S A S S T V G L S V S R R Q F L I K A G - - - L A S M A GG T L A L F GC H R A P L Q H F H G - - - - T M GG R F D L GP R T T P K L GNWQ D L Y R Q RW T W
EbdA-like D.oleovorans Hxd3 - - - - - - - - - - - - - - M K E V K I S R R T F L K G T S A T V A L L S L N S L G F L G GN T I A N A T E K I - - - - - - - - - - - - - - - F E DWK Y A GW E N L H R E EW T W
EbdhA A.aromaticum - M T R D EM I S V E P E A A E L Q D Q H R R D F L K R S G A A V L S L S L S S L A T G V V P G F L K D A QA G - - - - - - - - - - - - - - - T K A P G Y A S W E D I Y R K EWKW
EbdhA2 A.aromaticum M D D L K N T D A I R T GV S S A F D Q N R R G F L K R S G A G A L S L S L S S F A A G L V P G F V N A A QA G - - - - - - - - - - - - - - - K R G P T Y A T W E D V Y R N EWKW
EbdA-like gamma proteobachgter - - - - - M T L GA GM G I LWK Q K F D R R S F L K A S G - - - Y T V A A A A A V E L P - - - - - - S L H F K T - - - - - - - - A L A S D A A T P A P L K T W E D L Y R E RW T W
100 110 120 130 140 150 160 170 180. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
C25dhA S.denitrificans D R V V K G T H T R - A N C I G A C S WD V Y V K D G I AWR E E Q A A I Y E P H R P D I P D F N P R GC QK GA C Y T H L Q V N E S R I K Y P L K R V G E R G E GKWK R I T WD
peg.1060 S.denitrificans D K V V K G T H T R - A N C V A A C S WD V Y V R D G I AWR E E Q N T I Y E P P R P G I P D QN P R GC QK GA C Y T T L Q L S E T R V K Y P L K R V G E R G E GKWK R I T WD
peg.62 S.denitrificans D S V A K S T H F V N CW Y QR N C S WN V Y V K N G I AWR E E Q A A T Y E Q V D P N V P D Y N P R GC QK GA C Y S Q RM Y D A G R L T H P L R R V G A R G E GKWM R V S WD
peg.1309 S.denitrificans D K V V K G T H T R - A N C GD A C S WD V Y V R D G I AWR E E Q N A I Y E P H R A D V P DM N P R GC QK GA C Y T N L Q L S E A R L K Y P L K R V G E R G E GKWK R I S WD
peg.1642 S.denitrificans D K V A K G S H GW - A N C R S A C EWD L Y V K D GV V V R E E Q S A T Y E A S E P G I P D F N P R GC QK GA C Y T E V M Y G P S R T T V P L K R V G P R GS GKW E K I S W E
peg.481 S.denitrificans D K V A K G S H GW - A N C R S A C EWD L Y V K D GV V V R E E Q S A T Y E A S E P G I P D F N P R GC QK GA C Y T E V M Y G P S R T T V P L K R V G P R GS GKW E K I S W E
peg.761 S.denitrificans D R V V K G T H T R - A N C I A A C S WD V Y V R D G I AWR E E Q N A I Y E P H R P D I P D F N P R GC QK GA C Y T T L Q L S E A R L K Y P L K R I G K R G E GKWK R I T WD
peg.1646 S.denitrificans D K V A K G S H GW - A N C R S A C EWD L Y V K D G I V V R E E Q S A T Y E A S E P G V P D F N P R GC QK GA C Y T E V M Y G P S R T T V P L K R V G P R GS GKW E K I S W E
EbdA-like D.oleovorans Hxd3 D K V T Y G T H L V D C Y P - G N C L WR V Y S K D GV V F R E E Q A A K Y P V I D P S G P D F N P R GC QK GA S Y S L QM Y N P D R L K Y P M K Q V G GR GS GKWK R V S WD
EbdhA A.aromaticum D K V NWG S H L N I CWP QG S C K F Y V Y V R N G I V WR E E Q A A Q T P A C N V D Y V D Y N P L GC QK GS A F N N N L Y G D E R V K Y P L K R V G K R G E GKWK R V S WD
EbdhA2 A.aromaticum D K V TWG S H L N I CWP QG S C K F Y V Y V R N G I V WR E E Q A A Q T A A C N P D Y V D Y N P S GC QK GA A F N N N L Y G E E R L K Y P L K R V G K R G E GKWK R V S WD
EbdA-like gamma proteobachgter D R V V K S S H GW - L N C R S A C EWD I Y V K D GV V V R E E Q T A T Y E A S E P G I P D F N P R GC QK GA C Y T E V M Y G P S R L Y S P M K R V G E R GS GQW E K I S WD
190 200 210 220 230 240 250 260 270. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
C25dhA S.denitrificans E A L T E I A D K L I D A A V A E G T E S I I F D D G T T N A G Y G P E T A GD V R F A T S L Q T T K I D S WA G V S DM P M G L V Q T WGM Y N C E G T S D DW F R S D Y I V I W
peg.1060 S.denitrificans E A L N E I A D K L I D I S V E H G T E T I C F D D - L S N T G Y G P E T A GD F R F S T A L QV T R L D GWS G V G DM P L GV I Q T WGA F N C E G T S D DW F R S D Y I V I W
peg.62 S.denitrificans E A L A D I A D RM I D V M R T D G P G A I T WD P G T A N A G GG A S T A - P Y R L G F I L D T P M I D V N T E V G D H H Q GA QV T V GK I S F S GS M D D L F Y S D L I L V W
peg.1309 S.denitrificans E A L N E I C D K L I D V A I D QG T E S I I F D D G T T N GG F G P E T A GD V R F T E A L N C T QM D S WA G V S DM P M G L V Q T WGM F N S E GS A D DW YM S D F I V I W
peg.1642 S.denitrificans Q A L R E I A V K T V D A A E KWG T D T I Y Q D L GP N F D - F G A S T A GR F K F Q FM A GG I F A D NWA E I G D L N V GA S I T V GA A H L G GS A D EW F L S D F I V V W
peg.481 S.denitrificans Q A L R E I A V K T V D A A E KWG T D T I Y Q D L GP N F D - F G P S T G GR F K F Q F QV GG L F A D NWA E I G D L N I GA N I A L GA A H V G GS S D EW F L S D F I V V W
peg.761 S.denitrificans E A L T E I A D K L I D A A V A E G T E S I I F D D G T T N S G Y G P E T S GDWR F A D A I QA T K I D S WA G V S DM P M GA V Q T WGM Y N C E G T S D DW F R S D Y I V I W
peg.1646 S.denitrificans Q A L R E I A V K T V D A V E E Y G T D T V F Q D L GP N F D - F G P S T A GR F K F M Y QA S S L F S DMWG E I G D L N F GA T M A L GA A Q I G GS S D DW F L S D F I V V W
EbdA-like D.oleovorans Hxd3 Q C L A E I A E G I V D G L E A QG P E S I I F E S GP GN GG Y V H V M A - V H R L M V S L GA T V L D L D S T I G D F N R G I Y E T F GK F M FM D S V D GW Y F G K L L L I W
EbdhA A.aromaticum E A A G D I A D S I I D S F E A QG S D G F I L D A P H V H A G S I AWGA - G F RM T Y L M D G V S P D I N V D I G D T YM GA F H T F GKM HM G Y S A D N L L D A E L I F M T
EbdhA2 A.aromaticum E A T A D I A D A I I D G I E T E G T D S F I L D S P H V H A G S V A N S G - G Y RM T Y L L D G V S P D N N V D I G D T Y S GA F H T F GKM HM G Y S A D N L L D S E L I F M T
EbdA-like gamma proteobachgter Q A L G E I A E K I V D I S E K Y G T D Y I I H DM GP H H D - F G P T T A A R A R F F S M L GA S L A D DWA E I G D L N V A A T M T F G F P H V G GS S D EW F L S D Y L V V W
280 290 300 310 320 330 340 350 360. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
C25dhA S.denitrificans V GN P I Y T R I P E A H F L H E A R Y R G A K L V V I A P D L N P S T V H A D T W L K I N P E T D A A L G L A A A Q V M I T E N L I K K D Y V L E Q T DM P F L V R K D D K R F L
peg.1060 S.denitrificans L GN P N Y T R I P D A H F L H E A R Y R G A K L V V V S P D L N A S T V H A D RW I K V K P E T D A A L G L A C A Q V M I A E D L Y K K D Y V L E Q T D F P F L V R K D N QR F L
peg.62 S.denitrificans G A N P V Y T Q I P N A H F I N E A R Y N G A K V V S I A P D Y N A S S I H A D L W I G V N S GS D A A L G L S L A Q V I I E E K L H QP D F I R E Q T D L P L L V R E D N QQ Y L
peg.1309 S.denitrificans V GN P N Y T K I P E A H F F H E A R Y R G A KM C V I A P D L N P S S V H A DMWV K L R P E S D P A F G L A A A Q V I I E E K L Y K L D Y I L E Q T D F P F L V R K D N QR F L
peg.1642 S.denitrificans MM N P S V T Q I P D A H F L Y E A R Y N G T E L C V I D P Q Y S A T A I H A D QW L P L E S G T D A A L G L A V A R Y L L D T G A I D L P Y I R E Q T D L P L L A R L D T GR F L
peg.481 S.denitrificans MM N P S V T Q I P D A H F L Y E A R Y N G T E L C V V D P Q Y S A T A I H A D QW L P L E S G T D A A L G L A V A R H L F E V N A I D L P Y V R E Q T D L P L L A R L D T GR F L
peg.761 S.denitrificans V GN P I Y T R I P E A H F L H E A R Y R G A K L V V I A P D L N P S T V H A D T W I N I K P E T D A A L G L A A A Q V M I S E N L Y K Q D Y V L E Q T D F P F L V R K D N QR F L
peg.1646 S.denitrificans MM N P S V T Q I P D A H F L Y E A R Y N G T E L C V I D P Q Y S A T A I H A D QW L P I H T G T D A A L G L A V A R H L L E V G A I D L P F I R E Q T D L P L L V R I D S GR F L
EbdA-like D.oleovorans Hxd3 HM N P V Y T R I P S Y H F I S E A R Y N G A E I I S I A P D Y N P S CM H A D E Y I P V EM GS D A A L G L A V C Q V L M N K KWV D Y P F V K E Q S D L P L L V R K D T D R F L
EbdhA A.aromaticum C S NWS Y T Y P S S Y H F L S E A R Y K G A E V V V I A P D F N P T T P A A D L H V P V R V GS D A A F W L G L S Q V M I D E K L F D R Q F V C E Q T D L P L L V RM D T GK F L
EbdhA2 A.aromaticum C S NWS Y T Y P S S Y H F M T E A R Y K G A E V V V V A P D F N P T T P G A D L H V P V K V GR D A A F W L G L C Q V M I D E K L I D R Q F A S E Q T D L P L L V R T D N GK F L
EbdA-like gamma proteobachgter MM N P S V T Q I P D A H F L F E A K Y N G A T L T V I D P Q Y S A T A I H A D HWM P I E S G T D A A L AM Y V S R Y I W E N D R I D L P Y V K E Q T D L P M L V R I D N GR F L
370 380 390 400 410 420 430 440 450. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
C25dhA S.denitrificans R GA DM V K G GA D N A V Y I WD E A K Q A A V V A P GC E G D G E G GR S L K L N G I K P A L S G T F T V K L A N G E S V E V H T V F DM L K E K L D T E Y T P E L A E K V T G
peg.1060 S.denitrificans R T S D V V K G GV D N A F Y L WD E A K N A I V M A P GC E G D G N G GR S L K L GK L K P A L S G T R S V K L L D GS T V E C V T V F DM I R E R L D T E H T P E Q A A K I T G
peg.62 S.denitrificans R QR D L K K D GR E D V F Y V WD E K T R S L R Q A P - - - - - - - - QK S L A L GK L R P A L E G R Y QV T L P D GR K V F V Q T V F S R L R Q Q L D S RWK P E Q T A A T T G
peg.1309 S.denitrificans R A S D V V K G GD E N A L Y F WD E A T D KM V I A P GCM G D G D G GR S L K L GK L K P A L S G T R S V K L L D GS T V E C E T V F D K L K L K L D T E Y T P E Q S A K I A D
peg.1642 S.denitrificans R E S D L K A G GD E D Q L YMWH P Q K N A A V P A P GC L K N - - - T T R S L K L D F E P P I D G QWK I K L A N G E E V V V V P V G AM L K E H L - D P W T F E H A A QV T H
peg.481 S.denitrificans R E E D L K D G GS P D Q L YMWH P Q QN A P V P A P GC T G N - - - H T R K L T L D F E P P I D G QW T I K L K D G E E V A V V P I G AM L K E H L - E P W T F E H A A Q I T G
peg.761 S.denitrificans R A S D L V E G GA D N A L H V WD E A R N QA V I A P GC E G D G N G GR S L KM GG I K P A L S G T F S V K L V S G E T V E V Q T V F DM I K A R L D S E Y T P E Q A A R V T G
peg.1646 S.denitrificans R E E D L K D G GS P D Q L YMWH P Q K N A A V P A P GC T G N - - - T T R K L T L D F E P P I D G QW T I R L A N GQ E V L V A P V G A L L K E H L - E P W T F E H T A A V T K
EbdA-like D.oleovorans Hxd3 S A A D I E K G A R D D Q F C F WD S K N N K V V K A P - - - - - - - - L E T L K L - P C D P A L E G V Y K A T L L D GK T V E V E P V F N K L K A L L D S E Y T P E Q A S EM C R
EbdhA A.aromaticum S A E D V D - G G E A K Q F Y F F D E K A G S V R K A S - - - - - - - - R G T L K L - D FM P A L E G T F S A R L K N GK T I QV R T V F E G L R E H L - K D Y T P E K A S A K C G
EbdhA2 A.aromaticum S A A D V D - G GH A K Q F Y V I D E K S G A L R E A P - - - - - - - - R G T L R L - D G P V A L E G T F S A K L R D GG T V QV R P V F Q L M K D Q L D K E F T P E K A S A K S G
EbdA-like gamma proteobachgter T E Q D L Q K A GR T D V V Y YWD QN A G K P K L A S GS E G E L R H T R L H L D K D V D P A I E G I F QV QG A D GN V I H V T T V G S L I R E S L - E R Y T L D Y T A QV T K
S.denitrificans, S25dA3
S.denitrificans, EbdA-like
D.oleovorans, EbdA-like
S.denitrificans, S25dA
S.denitrificans, S25dA4
S.denitrificans, S25dA6
S.denitrificans, S25dA7
S.denitrificans, S25dA2
S.denitrificans, S25dA5
A.aromaticum, EbdA
strain HdN1, EbdA-like
A.aromaticum, EbdA2
HA CB CC CDB
10 20 30 40 50 60 70. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
C25dhA S.denitrificans - - - - - - - - - - - - - - - - - M Q I S R R Q F I V G S A V A A A G L G L Y S L R P K H Y V P A A P R P A D K - - - - - - - - - - - P G I
peg.1060 S.denitrificans - - - - - - - - - - - - - - - - - M Q V S R R H F I V G T A A V A A G A G L Y S L R P K L T G P K - P G I T M P - - - - - - - - - - - P L V
peg.62 S.denitrificans - - - - - - - - - - M E S K P GM I GM D R R S F L K - A GG S A L A L S L C H L E L L P M N AM A QA A GK D GN G N K A A A E L S P L E
peg.1309 S.denitrificans - - - - - - - - - - - - - - - - - M Q V S R R N F I V G S A V A A A G L G L Y S L K P K T P A A I K A G P A L P - - - - - - - - - - - P L V
peg.1642 S.denitrificans - - - - - - - - M G I L A T S N L V S A S R R K F L V M A G - - - M A S A A GA A V G L F G C S R A P L Q H F K G T - - - T A S G R F D L G
peg.481 S.denitrificans - - - - - - - - - M T T A S P A Q P N P A R R R F L I L A GK T T V A G I A A A A T G L P G C N RM P L Q H F H G - - - - T V D G R F D L G
peg.761 S.denitrificans - - - - - - - - - - - - - - M Q F M Q L T R R H F I M G S A A T V A G L A L Y S L R P R H Y V P A A P R P A D P - - - - - - - - - - - P T V
peg.1646 S.denitrificans - - - - - - M E R S S A S S T V G L S V S R R Q F L I K A G - - - L A S M A GG T L A L F G C H R A P L Q H F H G - - - - T M G G R F D L G
EbdA-like D.oleovorans Hxd3 - - - - - - - - - - - - - - M K E V K I S R R T F L K G T S A T V A L L S L N S L G F L GG N T I A N A T E K I - - - - - - - - - - - - - -
EbdhA A.aromaticum - M T R D EM I S V E P E A A E L QD Q H R R D F L K R S GA A V L S L S L S S L A T G V V P G F L K D A QA G - - - - - - - - - - - - - -
EbdhA2 A.aromaticum M D D L K N T D A I R T G V S S A F D Q N R R G F L K R S GA G A L S L S L S S F A A G L V P G F V N A A QA G - - - - - - - - - - - - - -
EbdA-like gamma proteobachgter - - - - - M T L GA GM G I L WK QK F D R R S F L K A S G - - - Y T V A A A A A V E L P - - - - - - S L H F K T - - - - - - - - A L A S D
80 90 100 110 120 130 140. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
C25dhA S.denitrificans P A K K V K Y N D Y S D I WR E KWKWD R V V K G T H T R - A N C I G A C S WD V Y V K D G I AWR E E QA A I Y E P H R P D I P D F N P
peg.1060 S.denitrificans P A K K V K Y N D Y S D I WR E KWKWD K V V K G T H T R - A N C V A A C S WD V Y V R D G I AWR E E QN T I Y E P P R P G I P D Q N P
peg.62 S.denitrificans A A A E L E Y R S F E D L Y R K KWHWD S V A K S T H F V N CW Y Q R N C S WN V Y V K N G I AWR E E QA A T Y E QV D P N V P D Y N P
peg.1309 S.denitrificans S A K K I R Y N D Y S D I WR E KWKWD K V V K G T H T R - A N C G D A C S WD V Y V R D G I AWR E E QN A I Y E P H R A D V P DM N P
peg.1642 S.denitrificans P R T T P K L G NWQ D L Y R Q RW T WD K V A K G S H GW - A N C R S A C EWD L Y V K D G V V V R E E QS A T Y E A S E P G I P D F N P
peg.481 S.denitrificans P R T T P K L N NWQ D L Y R Q RW T WD K V A K G S H GW - A N C R S A C EWD L Y V K D G V V V R E E QS A T Y E A S E P G I P D F N P
peg.761 S.denitrificans P A K K V K Y N D Y S D I WR E KWKWD R V V K G T H T R - A N C I A A C S WD V Y V R D G I AWR E E QN A I Y E P H R P D I P D F N P
peg.1646 S.denitrificans P R T T P K L G NWQ D L Y R Q RW T WD K V A K G S H GW - A N C R S A C EWD L Y V K D G I V V R E E QS A T Y E A S E P G V P D F N P
EbdA-like D.oleovorans Hxd3 - F E DWK Y A GW E N L H R E EW T WD K V T Y G T H L V D C Y P - G N C L WR V Y S K D G V V F R E E QA A K Y P V I D P S G P D F N P
EbdhA A.aromaticum - T K A P G Y A S W E D I Y R K EWKWD K V NWG S H L N I CWP Q G S C K F Y V Y V R N G I V WR E E QA A Q T P A C N V D Y V D Y N P
EbdhA2 A.aromaticum - K R G P T Y A TW E D V Y R N EWKWD K V T WG S H L N I CWP Q G S C K F Y V Y V R N G I V WR E E QA A Q T A A C N P D Y V D Y N P
EbdA-like gamma proteobachgter A A T P A P L K TW E D L Y R E RW T WD R V V K S S H GW - L N C R S A C EWD I Y V K D G V V V R E E Q T A T Y E A S E P G I P D F N P
150 160 170 180 190 200 210. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
C25dhA S.denitrificans R G C Q K GA C Y T H L Q V N E S R I K Y P L K R V G E R G E G KWK R I T WD E A L T E I A D K L I D A A V A E G T E S I I F D D G T T N
peg.1060 S.denitrificans R G C Q K GA C Y T T L Q L S E T R V K Y P L K R V G E R G E G KWK R I T WD E A L N E I A D K L I D I S V E H G T E T I C F D D - L S N
peg.62 S.denitrificans R G C Q K GA C Y S Q RM Y D A G R L T H P L R R V GA R G E G KWM R V S WD E A L A D I A D RM I D V M R T D G P GA I T WD P G T A N
peg.1309 S.denitrificans R G C Q K GA C Y T N L Q L S E A R L K Y P L K R V G E R G E G KWK R I S WD E A L N E I C D K L I D V A I D QG T E S I I F D D G T T N
peg.1642 S.denitrificans R G C Q K GA C Y T E V M Y GP S R T T V P L K R V GP R GS G KW E K I S W E Q A L R E I A V K T V D A A E KWG T D T I Y Q D L G P N F
peg.481 S.denitrificans R G C Q K GA C Y T E V M Y GP S R T T V P L K R V GP R GS G KW E K I S W E Q A L R E I A V K T V D A A E KWG T D T I Y Q D L G P N F
peg.761 S.denitrificans R G C Q K GA C Y T T L Q L S E A R L K Y P L K R I GK R G E G KWK R I T WD E A L T E I A D K L I D A A V A E G T E S I I F D D G T T N
peg.1646 S.denitrificans R G C Q K GA C Y T E V M Y GP S R T T V P L K R V GP R GS G KW E K I S W E Q A L R E I A V K T V D A V E E Y G T D T V F Q D L G P N F
EbdA-like D.oleovorans Hxd3 R G C Q K GA S Y S L QM Y N P D R L K Y P M K Q V GG R GS G KWK R V S WD Q C L A E I A E G I V D G L E A QG P E S I I F E S G P GN
EbdhA A.aromaticum L G C Q K GS A F N N N L Y GD E R V K Y P L K R V GK R G E G KWK R V S WD E A A G D I A D S I I D S F E A QG S D G F I L D A P H V H
EbdhA2 A.aromaticum S G C Q K GA A F N N N L Y G E E R L K Y P L K R V GK R G E G KWK R V S WD E A T A D I A D A I I D G I E T E G T D S F I L D S P H V H
EbdA-like gamma proteobachgter R G C Q K GA C Y T E V M Y GP S R L Y S P M K R V G E R GS G QW E K I S WD Q A L G E I A E K I V D I S E K Y G T D Y I I H DM G P H H
220 230 240 250 260 270 280. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
C25dhA S.denitrificans A G Y G P E T A GD V R F A T S L Q T T K I D S WA GV S DM P M G L V Q T WGM Y N C E G T S D DW F R S D Y I V I WV G N P I Y T R I P
peg.1060 S.denitrificans T G Y G P E T A GD F R F S T A L QV T R L D GWS GV G DM P L G V I Q T WG A F N C E G T S D DW F R S D Y I V I W L G N P N Y T R I P
peg.62 S.denitrificans A G GG A S T A - P Y R L G F I L D T P M I D V N T E V G D H H QG A Q V T V G K I S F S G S M D D L F Y S D L I L V WG A N P V Y T Q I P
peg.1309 S.denitrificans G G F G P E T A GD V R F T E A L N C T QM D S WA GV S DM P M G L V Q T WGM F N S E G S A D DW YM S D F I V I WV G N P N Y T K I P
peg.1642 S.denitrificans D - F G A S T A GR F K F Q F M A GG I F A D NWA E I G D L N V G A S I T V G A A H L GG S A D EW F L S D F I V V WMM N P S V T Q I P
peg.481 S.denitrificans D - F G P S T G GR F K F Q F Q V GG L F A D NWA E I G D L N I G A N I A L G A A H V GG S S D EW F L S D F I V V WMM N P S V T Q I P
peg.761 S.denitrificans S G Y G P E T S GDWR F A D A I QA T K I D S WA GV S DM P M G A V Q T WGM Y N C E G T S D DW F R S D Y I V I WV G N P I Y T R I P
peg.1646 S.denitrificans D - F G P S T A GR F K F M Y Q A S S L F S DMWG E I G D L N F G A TM A L G A A Q I GG S S D DW F L S D F I V V WMM N P S V T Q I P
EbdA-like D.oleovorans Hxd3 G G Y V H V M A - V H R L M V S L GA T V L D L D S T I G D F N R G I Y E T F G K F M F M D S V D GW Y F GK L L L I WHM N P V Y T R I P
EbdhA A.aromaticum A G S I AWG A - G F RM T Y L M D G V S P D I N V D I G D T YM G A F H T F G KM HM G Y S A D N L L D A E L I F M T C S NWS Y T Y P S
EbdhA2 A.aromaticum A G S V A N S G - G Y RM T Y L L D G V S P D N N V D I G D T Y S G A F H T F G KM HM G Y S A D N L L D S E L I F M T C S NWS Y T Y P S
EbdA-like gamma proteobachgter D - F G P T T A A R A R F F S M L GA S L A D DWA E I G D L N V A A TM T F G F P H V GG S S D EW F L S D Y L V V WMM N P S V T Q I P
290 300 310 320 330 340 350. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
C25dhA S.denitrificans E A H F L H E A R Y R G A K L V V I A P D L N P S T V H A D T W L K I N P E T D A A L G L A A A Q V M I T E N L I K K D Y V L E Q T DM P F
peg.1060 S.denitrificans D A H F L H E A R Y R G A K L V V V S P D L N A S T V H A D RW I K V K P E T D A A L G L A C A Q V M I A E D L Y K K D Y V L E Q T D F P F
peg.62 S.denitrificans N A H F I N E A R Y N G A K V V S I A P D Y N A S S I H A D L W I G V N S G S D A A L G L S L A Q V I I E E K L H Q P D F I R E Q T D L P L
peg.1309 S.denitrificans E A H F F H E A R Y R G A KM C V I A P D L N P S S V H A DMWV K L R P E S D P A F G L A A A Q V I I E E K L Y K L D Y I L E Q T D F P F
peg.1642 S.denitrificans D A H F L Y E A R Y N G T E L C V I D P Q Y S A T A I H A D QW L P L E S G T D A A L G L A V A R Y L L D T G A I D L P Y I R E Q T D L P L
peg.481 S.denitrificans D A H F L Y E A R Y N G T E L C V V D P Q Y S A T A I H A D QW L P L E S G T D A A L G L A V A R H L F E V N A I D L P Y V R E Q T D L P L
peg.761 S.denitrificans E A H F L H E A R Y R G A K L V V I A P D L N P S T V H A D T W I N I K P E T D A A L G L A A A Q V M I S E N L Y K Q D Y V L E Q T D F P F
peg.1646 S.denitrificans D A H F L Y E A R Y N G T E L C V I D P Q Y S A T A I H A D QW L P I H T G T D A A L G L A V A R H L L E V G A I D L P F I R E Q T D L P L
EbdA-like D.oleovorans Hxd3 S Y H F I S E A R Y N G A E I I S I A P D Y N P S CM H A D E Y I P V EM G S D A A L G L A V C Q V L M N K KWV D Y P F V K E Q S D L P L
EbdhA A.aromaticum S Y H F L S E A R Y K G A E V V V I A P D F N P T T P A A D L H V P V R V G S D A A F W L G L S Q V M I D E K L F D R Q F V C E Q T D L P L
EbdhA2 A.aromaticum S Y H F M T E A R Y K G A E V V V V A P D F N P T T P G A D L H V P V K V G R D A A F W L G L C Q V M I D E K L I D R Q F A S E Q T D L P L
EbdA-like gamma proteobachgter D A H F L F E A K Y N G A T L T V I D P Q Y S A T A I H A D HWM P I E S G T D A A L AM Y V S R Y I W E N D R I D L P Y V K E Q T D L P M
S.denitrificans, S25dA
S.denitrificans, S25dA3
S.denitrificans, EbdA-like
S.denitrificans, S25dA4
S.denitrificans, S25dA6
S.denitrificans, S25dA7
S.denitrificans, S25dA2
S.denitrificans, S25dA5
D.oleovorans, EbdA-like
A.aromaticum, EbdA
A.aromaticum, EbdA 2
strain HdN1, EbdA-like
A
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Fig. 6
FS IV
VACKTNKCIGCHTCS RICNHCTHPAC EACPR
MCFRFGRCRYPCAERACAQ-YCFICKQ GPCQR
FS IIIFS IIFS I
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Fig. 7
α3 (73%)S25dB (β)S25dC (γ)
S25dA (α)
α2 (82%)
γ7 (31%) β7 (59%) α7 (38%)
γ6 (31%) β6 (60%) α6 (38%)
γ5 (29%) β5 (59%) α5 (38%)
β4 (99%)γ4 (98%) α4 (71%) δ4
EbdC-like EbdB – like (59%) EbdA - like (41%) EbdD-like
(35%)
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Fig. 8
0.1
DmsA
BisC
TorA
DorA
TorA
PcrANarG NarG
ClrASerA
Type I
BisC
NapA / NasA FdnG
PhsA
PsrA
DdhA
γ-proteobacterium HdN1
S.denitrificans, S25dA2 Steroid C25
dehydrogenase
( like )
Ethylbenzene
dehydrogenase
( like )
Type III
Type II
DsrA
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Juri Dermer and Georg Fuchsatom in the side chain of cholesterol
Molybdoenzyme that catalyzes the anaerobic hydroxylation of tertiary carbon
published online September 1, 2012J. Biol. Chem.
10.1074/jbc.M112.407304Access the most updated version of this article at doi:
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