the cysteine regulatory complex from plants and microbes: what was old is new again

The cysteine regulatory complex from plants and microbes: whatwas old is new againJoseph M Jez and Sanghamitra Dey

Available online at www.sciencedirect.com

The physical organization of enzymes in metabolism is an old

concept being revisited by new experimental approaches. In

plants and microbes, the enzymes of cysteine biosynthesis —

serine acetyltransferase (SAT) and O-acetylserine sulfhydrylase

(OASS) — form a bi-enzyme complex called the cysteine

regulatory complex (CRC), which likely plays a role in

modulating cysteine biosynthesis in response to sulfur nutrient

state. Structural and biochemical studies of SAT and OASS as

individual enzymes and recent advances in structural,

biophysical, and in vivo analysis of the CRC provide new

insights on the function of this macromolecular assembly in

plants and microbes and opens biotechnology and

pharmaceutical opportunities for future exploration.

Address

Department of Biology, Washington University in St. Louis, One

Brookings Drive, Campus Box 1137, St. Louis, MO 63130, United States

Corresponding author: Jez, Joseph M ([email protected])

Current Opinion in Structural Biology 2013, 23:302–310

This review comes from a themed issue on Macromolecular

assemblies

Edited by Felix Rey and Wesley I Sundquist

For a complete overview see the Issue and the Editorial

Available online 17th March 2013

0959-440X/$ – see front matter, # 2013 Elsevier Ltd. All rights

reserved.

http://dx.doi.org/10.1016/j.sbi.2013.02.011

IntroductionMetabolic regulation in biochemical pathways is often

viewed as a series of controls that alter activity of key

proteins in response to changes in cellular environment.

Classically, these mechanisms include feedback inhi-

bition, allosteric effects, post-translational modifications,

and cellular localization. Biochemical regulation of

proteins is typically studied in isolated systems, but

proteins function in a crowded environment. This idea

harkens back to the concept of glycolytic enzymes form-

ing metabolons and the basis of how the organization of

enzymes into macromolecular complexes can influence

metabolism, often by channeling metabolites between

enzyme active sites [1–2]. In the era of synthetic biology,

the manipulation of biochemical organization and altera-

tion of physical localization of enzymes is rapidly becom-

ing part of the standard toolbox for enhancing the

production of molecules of interest [3–6]. Although


multi-enzyme complexes in metabolism are an emerging

area of interest, our understanding of these assemblies

remains limited. For example, one of the oldest examples

of these systems is only beginning to be understood.

More than 40 years ago, Tomkins and coworkers ident-

ified a multiprotein complex from Salmonella that con-

tained the two enzymes from cysteine biosynthesis [7�,8].

In plants and microbes, incorporation of sulfide into

cysteine requires serine acetyltransferase (SAT) and O-

acetylserine sulfhydrylase (OASS; also known as O-acet-

ylserine(thiol)lyase or cysteine synthase) (Figure 1). SAT

transfers acetate from acetyl-coenzyme-A (acetyl-CoA)

onto serine to yield O-acetylserine in the first reaction.

In the second step, OASS uses pyridoxal 50-phosphate

(PLP) to catalyze the formation of cysteine from O-

acetylserine and sulfide. Originally, the role of the

‘cysteine synthase complex’ was thought to be metabolic

channeling, but later kinetic studies showed this was not

the case [9], which has left both how SAT and OASS

interact and the physiological reason for complex for-

mation as open questions. Growing evidence from studies

of plant cysteine biosynthesis suggests that formation of

the complex plays a regulatory role in response to cellular

metabolism; hence, the suggested name ‘cysteine regu-

latory complex’ (CRC) to better reflect its physiological

role [10,11]. Here we review the progress in understand-

ing the structure and function of SAT and OASS both

individually and as components in the CRC and highlight

unanswered questions for this macromolecular assembly.

Serine acetyltransferaseMultiple X-ray crystal structures of bacterial SAT reveal

the molecular architecture for catalysis and feedback

inhibition of the first step in cysteine biosynthesis

[12�,13,14]. These structures demonstrate that the SAT

monomer consists of an N-terminal alpha-helical domain

and a C-terminal left-handed parallel b-helix domain. To

date, both hexameric and trimeric SAT have been

described in the literature [12�,13–15] and as unpublished

structures (PDB: 3GVD, 3MC4, 3F1X). The hexameric

SAT are a dimer of trimers associated through a head-to-

head orientation of the N-terminal domains. This pos-

itions the active sites at the ‘tail’ ends of the structure with

residues at the monomer–monomer interface contribut-

ing to formation of substrate binding site (Figure 2a).

Comparison of the CoA and cysteine bound complexes

indicates that cysteine binds in the same site as serine,

thus explaining the basis for feedback inhibition, and

reveals the conformational changes that occur between

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Cysteine regulatory complex Jez and Dey 303

Figure 1

acetyl-CoAsulfide

cysteineO-acetylserineserineSerine

Acetyltransferase (SAT)O-Acetylserine

Sulfhydrylase (OASS)

OH

CO2-

CoA

CO2-

S2- SH

CO2-+H3N+H3N+H3N

S O

O O

Current Opinion in Structural Biology

Cysteine biosynthesis reactions catalyzed by serine acetyltransferase

(SAT) and O-acetylserine sulfhydrylase (OASS) in plants and bacteria.

the CoA and inhibitor bound forms. Cysteine binding

alters the position of the C-terminal region to occlude

binding of acetyl-CoA at the active site (Figure 2b). The

Haemophilius influenzae SAT structure suggested that

His154 functions as a general base with His189 and

Asp139 contributing to catalysis (Figure 2c). Site-directed

mutagenesis and kinetic studies support a role for His154

as the acceptor of a proton from the b-hydroxyl of serine

as the tetrahedral intermediate is formed following

nucleophilic attack on the thioester carbonyl of acetyl-

CoA. His189 contributes to substrate binding and orien-

tation for efficient catalysis and Asp139 interacts with

His154 to enhance basicity [16].

Figure 2

(a)

(b)

CoA

cysteine

(OAsp139

His154

His189

His189

NHN

OH

S CoA

O-

ONH

N

+H3N

+H3N

+H3N

-O2C

-O2C CO2-

+N

N

OO-

S-CoA

SH-CoA

O

O

+

Reaction mechanism and structure of serine acetyltransferase (SAT). (a) Pro

SAT hexamer. Each monomer is colored differently. The location of the CoA

sites between monomers in the left-side trimer are indicated. The positions of

complex with each molecule individually. (c) Overlay of H. influenzae SAT�Cchanges in the 182–184 and C-terminal (241–257) loops (pink) that occur up


O-acetylserine sulfhydrylase and theb-substituted alanine synthase enzyme familyThe second enzyme in cysteine synthesis (i.e. OASS)

catalyzes the b-replacement of the acetoxy group of O-

acetylserine with sulfide to yield cysteine and acetate as

products (Figure 3a). The reaction chemistry is driven by

the presence of the pyridoxal 50-phosphate (PLP) cofactor

at the active site and uses a ping-pong mechanism that

yields a stable a-aminoacrylate reaction intermediate,

which is accessible to the sulfide nucleophile in the

second half-reaction [17].

Structurally, the bacterial and plant OASS function as

homodimers with one PLP per monomer (Figure 3b)

[18,19,20�]. Binding of O-acetylserine and reaction with

PLP positions the substrate to interact with residues in an

active site loop to trigger a conformational change that

results in a subdomain movement and enclosure of the

PLP-linked intermediate (Figure 3c) [18]. Site-directed

mutagenesis, kinetic analysis, and ligand binding exper-

iments indicate that Asn77 and Gln147 are key amino

acids for O-acetylserine binding and that Thr74 and Ser75

are involved in sulfur incorporation into cysteine [20�,21].

The domain movement also narrows the active site open-

ing to control sulfide access to the a-aminoacrylate reac-

tion intermediate in the second half-reaction.

The active site entrance varies in size between the two

bacterial OASS isoforms — CysK and CysM. CysK is the

CoA

cys

resi241-257

resi182-184

c)


posed catalytic mechanism of SAT. (b) Overall structure of the bacterial

(blue space-filling model) and cysteine (tan space-filling model) binding

the two ligands are derived from crystal structures of H. influenzae SAT in

oA (tan) and SAT�cysteine (olive) complexes showing the structural

on cysteine binding.


304 Macromolecular assemblies

Figure 3

(a) (b)

(c)

Q147T74

T78S75

G76

N77A46

(d)

O

OCO2

-

NH2

CO2-

O-

O-

O-

O-

O4P

O4P

O4P

O4P

+

NH

NH

+NH

HS

cysteine

-SH

HN

+NH

CO2-

CO2-

CO2-

N+HO-

N

2-O4P

N+

N+

N+H

H

acetate

H

O

O

+

Lys

pyruvate

cysteine

cysteine

S-sulfocysteineacetate

acetate

thiosulfate

cysteinephosphate

O-phosphoserine

O-acetylserine

α-aminoacrylate

β-cyanoalanine

HS-

HS-HS-

HS-CN-CAS CAS

DES

DES

OASSOASS

OPSS

OPSS

SSCS/CysM

SSCS/CysM


Structure and function of O-acetylserine sulfhydrylase (OASS) and the b-substituted alanine synthase (BSAS) family of enzymes. (a) A simplified

reaction sequence for cysteine formation catalyzed by OASS is shown. Pyridoxal 50-phosphate (PLP) forms a Schiff base with an active site lysine.

Addition of O-acetylserine in the first half reaction results in loss of acetate and formation of a stable a-aminoacrylate reaction intermediate. In the

second half reaction, nucleophilic attack of sulfide results in cysteine production. (b) Structure of the A. thaliana OASS homodimer is shown as ribbon

diagram. The left-hand monomer shows secondary structure features with a-helices and b-strands colored rose and blue, respectively. The location of

Lys46 and PLP are shown as stick models (gold). (c) Active site view of the A. thaliana OASS K46A mutant. Removal of the active site lysine allows for

trapping of PLP linked to methionine as an external aldimine mimic of substrate binding. (d) Biosynthetic diversity in the BSAS family of enzymes

results from substrate variability in the first (left-side) and second (right-side) half-reactions catalyzed by OASS (tan), S-sulfocysteine synthase (SSCS)/

CysM (green), b-cyanoalanine synthase (CAS; orange), cysteine desulfhydrase (DES, purple), and O-phosphoserine sulfhydrylase (OPSS, pink).

predominant form for cysteine biosynthesis and is highly

specific for sulfide as the nucleophile. CysM is expressed

under anaerobic conditions and accepts a wide range of

nucleophiles, including thiosulfate, for the second half-

reaction. Structural studies of CysM from Escherichia colirevealed amino acid variations that widen the active site

entrance to allow for productive binding of a broader

range of nucleophiles [22–25]. In addition, spectroscopic

studies indicate that the two isozymes display different

equilibrium of PLP tautomers because of greater confor-

mational flexibility in the CysM forms, which may also

affect reaction efficiency with various substrates [24]. The

specificity of OASS and related enzymes for substrates in

each half-reaction can lead to new biological function.


The inherent versatility of PLP-dependent chemistry

and the OASS scaffold provides a platform for the diver-

sification of activity across the b-substituted alanine

synthase (BSAS) enzyme family (Figure 3d) [26,27]. In

diverse plant species, b-cyanoalanine synthase is critical

for the detoxification of cyanide generated as a byproduct

during ethylene biosynthesis and uses cysteine and cya-

nide as substrates to produce b-cyanoalanine and sulfide

[26]. Recent structural studies of the b-cyanoalanine

synthase from soybean had identified critical residues

for substrate specificity in the first half-reaction of the

enzyme [28]. A BSAS family member in Arabidopsisthaliana (thale cress) was recently shown to encode a

cysteine desulfhydrase, which catalyzes the degradation



of cysteine to pyruvate, ammonia, and sulfide [29]. Sim-

ilarly, S-sulfocysteine synthase, the plant homolog of the

bacterial CysM, converts O-acetylserine and thiosulfate

into acetate and S-sulfocysteine, which can be metab-

olized back to cysteine and sulfate by reductive conver-

sion [30]. In the hyperthermophilic archaea Aeropyrumpernix, O-phosphoserine sulfhydrylase catalyzes a novel

cysteine synthetic reaction from O-phosphoserine and

sulfide [31–33]. The same reaction catalyzed by a CysM

in Mycobacterium tuberculosis appears to support an alter-

nate cysteine biosynthesis pathway in this pathogen [34].

At the biochemical level, the evolution of BSAS enzyme

function leads to changes in binding of the first substrate,

formation of a reactive a-aminoacrylate intermediate, and

nucleophilic attack by the second substrate to generate a

range of useful metabolic functions.

The cysteine regulatory complex in plants andbacteriaA central feature of cysteine biosynthesis in plants and

bacteria is the formation of a macromolecular complex

containing both SAT and OASS. Although the individual

enzymes in the assembly are well understood at the

biochemical level, recent examinations of CRC for-

mation, the effects on SAT and OASS in the complex,

and the role of this macromolecular complex in plants and

bacteria are shedding new light on the biological function

of the CRC.

Formation of the CRC in the plants and bacteria has been

demonstrated by a range of analytical methods, including

size-exclusion chromatography [35–37], yeast two-hybrid

[38–40], UV/vis spectroscopy [41�], surface plasmon

resonance [42], and isothermal titration calorimetry

[43�,44–46]. Multiple protein–protein interaction studies

have identified the C-terminal residues of SAT as essen-

tial for interaction with OASS through either deletion

analysis or mutagenesis [35–40,41�,42,43�,44–46].

The molecular basis for how OASS associates with SAT

was unclear until initial biochemical studies suggested

that the OASS active site provided a binding site for SAT

[20�,41�]. The X-ray crystal structures of the OASS from

H. influenzae and A. thaliana in complex with peptides

corresponding to the C-terminal ten amino acids of their

cognate SAT have confirmed that the OASS active site

serves as an anchor point for SAT binding (Figure 4a,b)

[43�,47�]. Multiple binding interactions between residues

in the OASS active site and the highly conserved C-

terminal isoleucine residue of SAT lock the tail region

of SAT in the active site cleft and are critical for nano-

molar binding affinity, as demonstrated by site-directed

mutagenesis studies [43�,44,45]. These structures also

explain the effect CRC formation on OASS activity.

The earliest studies of the plant CRC complex showed

that SAT activity was enhanced, whereas, the catalytic


efficiency of OASS was dramatically decreased in the

bienzyme complex [35]. These observations mirrored

those reported in the original isolation of the complex

from bacteria [7�]. In addition, accumulation of O-acet-

ylserine triggers the dissociation of the CRC [35]. The

crystal structures of OASS in complex with the C-term-

inal peptides of SAT readily explain the loss of OASS

activity in the CRC due to an occluded active site and the

dissociation of the complex from competition by O-acet-

ylserine [43�,47�].

Currently, it is unclear how CRC formation alters the

structure of SAT; however, it is likely that interaction

with OASS reorganizes the C-terminal region near the

active site, since association of the two enzymes in the

complex alters both the catalytic properties and protein

stability of SAT. Kinetic analysis of the soybean CRC

reveals that SAT shows a loss of substrate inhibition by

serine and a decrease in the effectiveness of feedback

inhibition by cysteine, along with improved catalytic

efficiency [45,46]. In bacteria, free SAT is more sensitive

to cysteine inhibition than the plant SAT and is not

altered by formation of the CRC [46]. Moreover, the

physical stability of the bacterial and plant SAT is

enhanced upon CRC formation [35–37,45].

Biophysical studies of the bacterial and plant CRC

demonstrate that the tight interaction of SAT and OASS

is driven by rapid association and extremely slow dis-

sociation [41�,42,43�,44–46,47�,48,49,50�]; however,

different models for the formation and composition of

the CRC are suggested. Analysis of soybean CRC for-

mation by isothermal titration calorimetry and surface

plasmon resonance suggested negative cooperativity as

OASS bound to SAT in three distinct binding events [45];

however, studies of the Arabidopsis CRC showed only a

single binding isotherm [46]. Computational modeling of

the plant CRC suggests that multiple OASS dimers can

bind to the trimeric ends of SAT with the first binding

event energetically preferred [50�]. Stopped-flow fluor-

escence spectroscopy of the CRC from H. influenzae and

E. coli agree with rapid formation of the complex, but

differ in the number of isomerization steps (i.e. two versus

three) during association and interpretation of the

observed changes [48,49]. In the H. influenzae CRC,

the conformational change that follows bienzyme associ-

ation may result from closure of the OASS active site

following binding of SAT [48], which would be consistent

with both binding studies [44] and simulations of the

Arabidopsis CRC [51] suggesting that binding at one

active site in the OASS homodimer alters the confor-

mation at the second active site to prevent substrate

binding. An alternate model from the E. coli CRC

suggests an initial non-allosteric interaction that anchors

the two enzymes together followed by additional confor-

mational changes that inactivate OASS [49]. The oligo-

merization of the CRC in plants and bacteria as a



Figure 4

I226

Sulfur Sufficient

(c)

CRC

CRC

OASSOASS

OASSOASSOASS

SAT

SATO-acetylserine

free+ sulfide

cysteine

enhanced SATactivity

•

•

•

•

release from

increasedstability

inhibition

inactivation ofOASS by gating

Sulfur Depletion

elevatedO-acetylserine

OASS + SAT

dissociationof CRC

activation of sulfateuptake & sulfur assimilation

PLPK46

Q147

Y V I*W

ES D

T S75T74

M125-K126

(a) (b)

active site


The cysteine regulatory complex (CRC) — initial structures and a model for biological function. (a) Binding of the C-terminal serine acetyltransferase

(SAT) peptide (rose) in the active site of A. thaliana O-acetylserine sulfhydrylase (OASS). The molecular surface of OASS is shown with the surface

corresponding to pyridoxal-50-phosphate (PLP) shown in yellow. (b) Binding interactions with the SAT peptide at the OASS active site are shown.

Interactions with the C-terminal isoleucine residue mimic the contacts made with substrate (see Figure 3c). Variability in the C-terminal regions and

conservation of the terminal isoleucine residue of SAT from A. thaliana (AtSAT), soybean (GmSAT), E. coli (EcSAT), and H. influenzae (HiSAT) are shown

in the inset. (c) Current model for the plant CRC under sulfur sufficient and depleted conditions. As part of the CRC (yellow box), OASS is inactive and

SAT-activated.

hexameric SAT in complex with two OASS dimers is

largely based on size-exclusion chromatography [7�,35];

however, different compositions of the CRC from Arabi-dopsis and soybean have been proposed based on analyti-

cal ultracentrifugation studies. In the model for

Arabidopsis, the oligomerization resembles that of the

bacterial CRC [46], whereas, another study using the

complex from soybean suggests that a trimeric SAT

can bind up to three OASS dimers [45]. The oligomer-

ization of the soybean SAT can vary between hexameric

and trimeric forms under various experimental con-

ditions, which may explain the different observations.

Moreover, there may be different effects depending on invitro and in vivo environments. As noted by compu-

tational studies, each C-terminal tail of the SAT trimeric

structure can bind an OASS homodimer, although

the addition of multiple molecules is energetically


unfavorable, which agrees with the observed experimen-

tal data [45,50�].

A model for biological function of the cysteineregulatory complex in plantsAlthough the CRC in plants and bacteria share common

features, including formation of the initial encounter

complex by binding of the C-terminal tail of SAT at

one active site in the OASS homodimer, structural

changes in the unbound active site that prevents catalysis,

and changes in the kinetic properties and stability of SAT

in the bienzyme complex, the biological role of this

macromolecular assembly is better understood in plants

than in bacteria (Figure 4c).

In plants, the role of the CRC appears to be for fine-

tuning of sulfur metabolism in response to nutrient levels



[52]. Under sulfur sufficient conditions, SAT and OASS

form the CRC, which enhances the catalytic efficiency of

SAT and leads to increased formation of O-acetylserine.

An excess of OASS catalyzes cysteine formation from

O-acetylserine and sulfide, provided by the sulfur assim-

ilation pathway. If sulfur supply becomes limiting,

accumulation of O-acetylserine leads to dissociation of

the CRC. Elevated O-acetylserine also increases expres-

sion of genes encoding sulfate transporters and enzymes

in the sulfur assimilation pathway to increase sulfate

update and metabolism to restore cellular sulfur con-

ditions that favor formation of the CRC and cysteine

synthesis [53–55]. In this system, the equilibrium of

uncomplexed and complexed SAT modulates the limit-

ing step in cysteine biosynthesis depending on sulfur

status. More recent in vivo studies also better define

the role of the CRC in the context of organelles in the

model plant Arabidopsis [56–59]. These studies suggest

that the mitochondria provides O-acetylserine and is the

likely site of flux control, that plastids are responsible for

the sulfur assimilation pathway and sulfide synthesis, and

that the cytosol is where cysteine synthesis occurs.

Unanswered questions and new directionsStructural, functional, and biological experiments are

providing new insight on the CRC, but many questions

remain unanswered and promise exciting future explora-

tions. For example, what is the molecular structure of the

CRC? Initial crystallographic studies of the plant and

bacterial OASS in complex with peptides corresponding

to the C-terminal tail of their cognate SAT yielded the

first glimpses of how the bienzyme complex is formed

[43�,47�]; however, the architecture of a fully assembled

CRC from either plants or microbes and how formation of

the complex alters structure of SAT and/or OASS

remains to be determined. Similarly, the basis for

changes in the biochemical properties of SAT following

interaction with OASS are poorly understood. At present,

structural details of the CRC are unavailable, but

approaches such as cryo-electron microscopy and small

angle X-ray scattering could be alternatives to X-ray

crystallography to develop low-resolution views of the

complex that help define similarities and differences

between this macromolecular assembly from various

plant and bacterial species [60–62].

Recent studies also raise the possibility of other protein

complexes related to cysteine metabolism, especially in

microbes. For example, in M. tuberculosis a protein com-

plex formed by CysM (i.e. OASS) and CysO, a sulfur

carrier protein, provides a new route for cysteine synthesis

[63�]. In this case, the reaction catalyzed by CysM uses a

thiocarboxylate of CysO for the replacement of the acetyl

group of O-acetylserine to yield cysteine. In addition,

reports that OASS stimulates activity of ATP sulfurylase,

an enzyme in sulfur assimilation, suggest the possibility of

other protein-protein interactions [64].


What is the physiological function of the CRC in bacteria?

Extensive work in plants suggests a role for this macro-

molecular assembly as a sensor of sulfur nutrient state in

cells and that the interplay of metabolism in different

organelles is critical for balanced control of fluxes [52–55].

Surprisingly, examinations of bacterial cysteine synthesis

and the contribution of the CRC to pathway regulation

have lagged behind the plant field. Future studies of

cysteine synthesis in bacteria that combine metabolic flux

analysis and pathway engineering may provide clues on

the function of this system.

With regard to studies of the plant CRC, nearly all

investigations of the complex have been performed in

the model plant Arabidopsis; however, sulfur nutrient

demands vary widely across plant species and how the

CRC functions may vary in different plants. Moreover,

the relevance of understanding the control of thiol metab-

olism in plants and microbes may have biotechnology

applications.

As an essential nutrient, improved sulfur uptake and

metabolism into sulfur-rich compounds has applications

for meeting the challenges of reducing fertilizer appli-

cation, enabling sustainable agriculture, and generating

low-input crops with increased performance in areas of

marginal soil quality [65]. Major crops such as corn,

soybean, and rice, contain low levels of sulfur-rich amino

acids. Thus, improvements in sulfur metabolism may be

valuable to the livestock and poultry industry by provid-

ing enhanced sulfur-containing amino acid content in

crops for improved nutritional value in animal feeds.

Modifying sulfur metabolism in crops may also lead to

new approaches for improving tolerance to abiotic stres-

ses, which are leading causes of crop yield reduction

[66,67]. In crop plants, cysteine biosynthesis contributes

critical metabolites for the synthesis of glutathione, which

is used to maintain redox balance in cells under abiotic

stress resulting from cold temperatures and other environ-

mental stresses. Modification of the regulation of thiol

metabolism to respond to abiotic stresses may aid in the

development of crops with improved tolerance to

environmental changes.

There is also a renewed interest in the enzymes of

cysteine biosynthesis from pathogenic microbes and pro-

tozoa as a possible pharmaceutical target. In addition to

work on the enzymes from Mycobacterium, recent studies

suggest that OASS in the pathogenic organisms Leishma-nia major, L. donovani, and Entamoeba histolytica is critical

for synthesis of glutathione and trypanothione, which

maintain redox homeostasis and provide protection

against oxidative stresses. Structural studies show that

the OASS in these protozoans share similar structural

features as homologs from bacterial and plant sources,

but also significant differences in active site structures,

and provide information useful for inhibitor development



[68–70]. Moreover, efforts to identify pharmacophores

and design peptidomimetic compounds based on inter-

action of SAT and OASS show promise [71,72].

References and recommended readingPapers of particular interest, published within the period of review,have been highlighted as:

� of special interest

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19. Burkhard P, Tai CH, Ristroph CM, Cook PF, Jansonius JN: Ligandbinding induces a large conformational change in O-acetylserine sulfhydrylase from Salmonella typhimurium. JMol Biol 1999, 291:941-953.

20.�

Bonner ER, Cahoon RE, Knapke SM, Jez JM: Molecular basis ofcysteine biosynthesis in plants: structural and functionalanalysis of O-acetylserine sulfhydrylase from Arabidopsisthaliana. J Biol Chem 2005, 280:38803-38813.

This article, along with Ref. [41�], provides the first experimental evidencefor the role of the plant OASS active site as the interaction site for the C-terminal tail of SAT in formation of the CRC. In this paper, mutants ofOASS generated from structural analysis of a highly conserved surfaceloop were used in pull-down assays to demonstrate complex formation.

21. Banerjee S, Ekka MK, Kumaran S: Comparative thermodynamicstudies on substrate and product binding of O-acetylserinesulfhydrylase reveals two different ligand recognition modes.BMC Biochem 2011, 12:31.

22. Claus MT, Zocher GE, Maier TH, Schulz GE: Structure of the O-acetylserine sulfhydrylase isoenzyme CysM from Escherichiacoli. Biochemistry 2005, 44:8620-8626.

23. Zocher G, Wiesand U, Schulz GE: High resolution structure andcatalysis of O-acetylserine sulfhydrylase isozyme B fromEscherichia coli. FEBS J 2007, 274:5382-5389.

24. Chattopadhyay A, Meier M, Ivaninskii S, Burkhard P, Speroni F,Campanini B, Bettati S, Mozzarelli A, Rabeh WM, Li L, Cook PF:Structure, mechanism, and conformational dynamics of O-acetylserine sulfhydrylase from Salmonella typhimurium:comparison of A and B isozymes. Biochemistry 2007,46:8315-8330.

25. Schnell R, Oehlmann W, Singh M, Schneider G: Structuralinsights into catalysis and inhibition of O-acetylserinesulfhydrylase from Mycobacterium tuberculosis: crystalstructures of the enzyme alpha-aminoacrylate intermediateand an enzyme-inhibitor complex. J Biol Chem 2007,282:23473-23481.

26. Watanabe M, Kusano M, Oikawa A, Fukushima A, Noji M, Saito K:Physiological roles of the beta-substituted alanine synthasegene family in Arabidopsis. Plant Physiol 2008,146:310-320.

27. Yi H, Jez JM: Assessing functional diversity in the soybean b-substituted alanine synthase enzyme family. Phytochemistry2012, 83:15-24.

28. Yi H, Juergens M, Jez JM: Structure of soybean b-cyanoalaninesynthase and the molecular basis for cyanide detoxification inplants. Plant Cell 2012, 24:2696-2706.

29. Alvarez C, Calo L, Romero LC, Garcıa I, Gotor C: An O-acetylserine(thiol)lyase homolog with L-cysteinedesulfhydrase activity regulates cysteine homeostasis inArabidopsis. Plant Physiol 2010, 152:656-669.

30. Bermudez MA, Paez-Ochoa MA, Gotor C, Romero LC:Arabidopsis S-sulfocysteine synthase activity is essential forchloroplast function and long-day light-dependent redoxcontrol. Plant Cell 2010, 22:403-416.

31. Mino K, Ishikawa K: A novel O-phospho-L-serine sulfhydrylationreaction catalyzed by O-acetylserine sulfhydrylase fromAeropyrum pernix K1. FEBS Lett 2003, 551:133-138.

32. Oda Y, Mino K, Ishikawa K, Ataka M: Three-dimensionalstructure of a new enzyme, O-phosphoserine sulfhydrylase,involved in I-cysteine biosynthesis by a hyperthermophilicarchaeon, Aeropyrum pernix K1, at 2.0 A resolution. J Mol Biol2005, 351:334-344.



33. Nakamura T, Kawai Y, Kunimoto K, Iwasaki Y, Nishii K, Kataoka M,Ishikawa K: Structural analysis of the substrate recognitionmechanism in O-phosphoserine sulfhydrylase from thehyperthermophilic archaeon Aeropyrum pernix K1. J Mol Biol2012, 422:33-44.

34. Agren D, Schnell R, Oehlmann W, Singh M, Schneider G: Cysteinesynthase (CysM) of Mycobacterium tuberculosis is an O-phosphoserine sulfhydrylase: evidence for an alternativecysteine biosynthesis pathway in mycobacteria. J Biol Chem2008, 283:31567-31574.

35. Droux M, Ruffet ML, Douce R, Job D: Interactions betweenserine acetyltransferase and O-acetylserine (thiol) lyase inhigher plants — structural and kinetic properties of the freeand bound enzymes. Eur J Biochem 1998, 255:235-245.

36. Mino K, Yamanoue T, Sakiyama T, Eisaki N, Matsuyama A,Nakanishi K: Effects of bienzyme complex formation ofcysteine synthetase from Escherichia coli on some propertiesand kinetics. Biosci Biotechnol Biochem 2000, 64:1628-1640.

37. Mino K, Imamura K, Sakiyama T, Eisaki N, Matsuyama A,Nakanishi K: Increase in the stability of serineacetyltransferase from Escherichia coli against coldinactivation and proteolysis by forming a bienzyme complex.Biosci Biotechnol Biochem 2001, 65:865-874.

38. Bogdanova N, Hell R: Cysteine synthesis in plants: protein-protein interactions of serine acetyltransferase fromArabidopsis thaliana. Plant J 1997, 11:251-262.

39. Wirtz M, Berkowitz O, Droux M, Hell R: The cysteine synthasecomplex from plants — mitochondrial serineacetyltransferase from Arabidopsis thaliana carries abifunctional domain for catalysis and protein-proteininteraction. Eur J Biochem 2001, 268:686-693.

40. Liszewska F, Lewandowska M, Płochocka D, Sirko A: Mutationalanalysis of O-acetylserine (thiol) lyase conducted in yeast two-hybrid system. Biochim Biophys Acta 2007, 1774:450-455.

41.�

Campanini B, Speroni F, Salsi E, Cook PF, Roderick SL, Huang B,Bettati S, Mozzarelli A: Interaction of serine acetyltransferasewith O-acetylserine sulfhydrylase active site: evidence fromfluorescence spectroscopy. Protein Sci 2005, 14:2115-2124.

Along with Ref. [20�], this article provides the first experimental evidencefor the role of the bacterial OASS active site as the interaction site for theC-terminal tail of SAT in formation of the CRC. Using a peptide corre-sponding to the C-terminal tail of SAT, the authors demonstrate inter-action with OASS by changes in fluorescence of PLP.

42. Berkowitz O, Wirtz M, Wolf A, Kuhlmann J, Hell R: Use ofbiomolecular interaction analysis to elucidate the regulatorymechanism of the cysteine synthase complex fromArabidopsis thaliana. J Biol Chem 2002, 277:30629-30634.

43.�

Francois JA, Kumaran S, Jez JM: Structural basis for interactionof O-acetylserine sulfhydrylase and serine acetyltransferasein the Arabidopsis cysteine synthase complex. Plant Cell 2006,18:3647-3655.

Crystallographic and biochemical analyses of the interaction of a C-terminal peptide of plant SAT with OASS revealed the molecular basisfor inactivation of OASS as part of the bi-enzyme complex. This workcomplements similar studies with a bacterial SAT/OASS system [47�].

44. Kumaran S, Jez JM: Thermodynamics of the interactionbetween O-acetylserine sulfhydrylase and the C-terminus ofserine acetyltransferase. Biochemistry 2007, 46:5586-5594.

45. Kumaran S, Yi H, Krishnan HB, Jez JM: Assembly of the cysteinesynthase complex and the regulatory role of protein-proteininteractions. J Biol Chem 2009, 284:10268-10275.

46. Wirtz M, Birke H, Heeg C, Muller C, Hosp F, Throm C, Konig S,Feldman-Salit A, Rippe K, Petersen G, Wade RC, Rybin V,Scheffzek K, Hell R: Structure and function of the hetero-oligomeric cysteine synthase complex in plants. J Biol Chem2010, 285:32810-32817.

47.�

Huang B, Vetting MW, Roderick SL: The active site of O-acetylserine sulfhydrylase is the anchor point for bienzymecomplex formation with serine acetyltransferase. J Bacteriol2005, 187:3201-3205.

This article along with Ref. [43�] directly showed how SAT interacts withOASS to form a bi-enzyme complex.


48. Salsi E, Campanini B, Bettati S, Raboni S, Roderick SL, Cook PF,Mozzarelli A: A two-step process controls the formation of thebienzyme cysteine synthase complex. J Biol Chem 2010,285:12813-12822.

49. Wang T, Leyh TS: Three-stage assembly of the cysteinesynthase complex from Escherichia coli. J Biol Chem 2012,287:4360-4367.

50.�

Feldman-Salit A, Wirtz M, Hell R, Wade RC: A mechanistic modelof the cysteine synthase complex. J Mol Biol 2009,386:37-59.

Using structural and biochemical studies of the plant and bacterial CRC, acomputational modeling approach is used to model formation of an intactCRC and suggested additional interaction regions.

51. Feldman-Salit A, Wirtz M, Lenherr ED, Throm C, Hothorn M,Scheffzek K, Hell R, Wade RC: Allosterically gated enzymedynamics in the cysteine synthase complex regulate cysteinebiosynthesis in Arabidopsis thaliana. Structure 2012,20:292-302.

52. Hell R, Hillebrand H: Plant concepts for mineral acquisition andallocation. Curr Opin Biotechnol 2001, 12:161-168.

53. Hirai MY, Fujiwara T, Awazuhara M, Kimura T, Noji M, Saito K:Global expression profiling of sulfur-starved Arabidopsis byDNA macroarray reveals the role of O-acetyl-L-serine as ageneral regulator of gene expression in response to sulfurnutrition. Plant J 2003, 33:651-663.

54. Hopkins L, Parmar S, Blaszczyk A, Hesse H, Hoefgen R,Hawkesford MJ: O-acetylserine and the regulation ofexpression of genes encoding components for sulfate uptakeand assimilation in potato. Plant Physiol 2005,138:433-440.

55. Maruyama-Nakashita A, Nakamura Y, Tohge T, Saito K,Takahashi H: Arabidopsis SLIM1 is a central transcriptionalregulator of plant sulfur response and metabolism. Plant Cell2006, 18:3235-3251.

56. Wirtz M, Hell R: Dominant-negative modification reveals theregulatory function of the multimeric cysteine synthaseprotein complex in transgenic tobacco. Plant Cell 2007,19:625-639.

57. Haas FH, Heeg C, Queiroz R, Bauer A, Wirtz M, Hell R:Mitochondrial serine acetyltransferase functions as apacemaker of cysteine synthesis in plant cells. Plant Physiol2008, 148:1055-1067.

58. Krueger S, Niehl A, Lopez Martin MC, Steinhauser D, Donath A,Hildebrandt T, Romero LC, Hoefgen R, Gotor C, Hesse H: Analysisof cytosolic and plastidic serine acetyltransferasemutants and subcellular metabolite distributions suggestsinterplay of the cellular compartments for cysteinebiosynthesis in Arabidopsis. Plant Cell Environ 2009,32:349-367.

59. Wirtz M, Beard KF, Lee CP, Boltz A, Schwarzlander M, Fuchs C,Meyer AJ, Heeg C, Sweetlove LJ, Ratcliffe RG, Hell R:Mitochondrial cysteine synthase complex regulates O-acetylserine biosynthesis in plants. J Biol Chem 2012,287:27941-27947.

60. Hindson VJ, Moody PC, Rowe AJ, Shaw WC: Serineacetyltransferase from Eschericia coli is a dimer of trimers. JBiol Chem 2000, 275:461-466.

61. Jonic S, Venien-Bryan C: Protein structure determination byelectron cryo-microscopy. Curr Opin Pharm 2009, 9:636-642.

62. Mertens HD, Svergun DI: Structural characterization of proteinsand complexes using small-angle X-ray solution scattering. JStruct Biol 2010, 172:128-141.

63.�

Jurgenson CT, Burns KE, Begley TP, Ealick SE: Crystal structureof a sulfur carrier protein complex found in the cysteinebiosynthetic pathway of Mycobacterium tuberculosis.Biochemistry 2008, 47:10354-10364.

This study, which presents the crystal structure of the CysM/OASS incomplex with the CysO sulfur carrier protein, demonstrates the structuralversatility of OASS and how protein complexes other than the CRC mayform using OASS as a foundation.



64. Wei J, Tang QX, Varlamova O, Roche C, Lee R, Leyh TS: Cysteinebiosynthetic enzymes are the pieces of a metabolic energypump. Biochemistry 2002, 41:8493-8498.

65. Rausch T, Wachter A: Sulfur metabolism — a versatile platformfor launching defence operations. Trends Plant Sci 2005,10:503-509.

66. Kim WS, Chronis D, Juergens M, Schroeder AC, Hyun SW, Jez JM,Krishnan HB: Transgenic soybean plants overexpressing O-acetylserine sulfhydrylase accumulate enhanced levels ofcysteine and Bowman–Birk protease inhibitor in seeds. Planta2012, 235:13-23.

67. Nguyen HC, Hoefgen R, Hesse H: Improving the nutritive valueof rice seeds — elevation of cysteine and methionine contentsin rice plants by ectopic expression of a bacterial serineacetyltransferase. J Exp Bot 2012, 63:5991-6001.

68. Raj I, Kumar S, Gourinath S: The narrow active-site cleft of O-acetylserine sulfhydrylase from Leishmania donovani allowscomplex formation with serine acetyltransferases with a rangeof C-terminal sequences. Acta Crystallogr D 2012, 68:909-919.


69. Fyfe PK, Westrop GD, Ramos T, Muller S, Coombs GH,Hunter WN: Structure of Leishmania major cysteine synthase.Acta Crystallogr F 2012, 68:738-743.

70. Chinthalapudi K, Kumar M, Kumar S, Jain S, Alam N, Gourinath S:Crystal structure of native O-acetyl-serine sulfhydrylase fromEntamoeba histolytica and its complex with cysteine:structural evidence for cysteine binding and lack ofinteractions with serine acetyl transferase. Proteins 2008,72:1222-1232.

71. Salsi E, Bayden AS, Spyrakis F, Amadasi A, Campanini B,Bettati S, Dodatko T, Cozzini P, Kellogg GE, Cook PF, Roderick SL,Mozzarelli A: Design of O-acetylserine sulfhydrylaseinhibitors by mimicking nature. J Med Chem 2010,53:345-356.

72. Spyrakis F, Felici P, Bayden AS, Salsi E, Miggiano R, Kellogg GE,Cozzini P, Cook PF, Mozzarelli A, Campanini B: Fine tuning of theactive site modulates specificity in the interaction of O-acetylserine sulfhydrylase isozymes with serineacetyltransferase. Biochim Biophys Acta 2013,1834:169-181.


the cysteine regulatory complex from plants and microbes: what was old is new again

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