1
Short Title: Dioxygenase chemistry by a diiron enzyme 1
2
TITLE: Castor Stearoyl-ACP Desaturase Can Synthesize a Vicinal Diol by Dioxygenase Chemistry. 3
Edward J. Whittle1, Yuanheng Cai1, Jantana Keereetaweep1, Jin Chai1, Peter H. Buist2 and John 4
Shanklin1*. 5
1Biology Department, Brookhaven National Laboratory, 50 Bell Avenue, Upton, NY 11973, USA. 6
2Department of Chemistry, Carleton University, 1125 Colonel By Drive, Ottawa, Ontario, Canada K1S 7
5B6. 8
* To whom correspondence should be addressed: [email protected], Tel. 631 344 3414, Fax. 631 344 9
6398. 10
One-sentence summary: The Ricinus communis stearoyl-ACP desaturase is capable of dioxygenase 11
chemistry, converting oleoly-ACP to the natural product erythro-9,10-dihydroxystearoyl-ACP. 12
13
Author contributions: JS, EJW and PHB designed the research; EJW performed the research; EJW, YC, JK 14
and JC contributed analytic/computational/ tools; EJW, YC, JC, PHB and JS analyzed the data; and EJW, JS 15
and PHB wrote the paper. 16
17
Plant Physiology Preview. Published on December 5, 2019, as DOI:10.1104/pp.19.01111
Copyright 2019 by the American Society of Plant Biologists
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ABSTRACT 18
In previous work, we identified a triple mutant of the castor (Ricinus communis) stearoyl-Acyl Carrier 19
Protein desaturase (T117R/G188L/D280K) that, in addition to introducing a double bond into stearate to 20
produce oleate, performed an additional round of oxidation to convert oleate to a trans allylic alcohol 21
acid. To determine the contributions of each mutation, in the present work we generated individual 22
castor desaturase mutants carrying residue changes corresponding to those in the triple mutant and 23
investigated their catalytic activities. We observed that T117R, and to a lesser extent D280K, 24
accumulated a novel product, namely erythro-9, 10-dihydroxystearate, that we identified via its methyl 25
ester through gas chromatography/mass spectrometry and comparison with authentic standards. The 26
use of 18O2 labeling showed that the oxygens of both hydroxyl moieties originate from molecular oxygen 27
rather than water. Incubation with an equimolar mixture of 18O2 and 16O2 demonstrated that both 28
hydroxyl oxygens originate from a single molecule of O2, proving the product is the result of dioxygenase 29
catalysis. Using prolonged incubation, we discovered that wild-type castor desaturase is also capable of 30
forming erythro-9, 10-dihydroxystearate, which presents a likely explanation for its accumulation to 31
approximately 0.7% in castor oil, of which the biosynthetic origin had remained enigmatic for decades. 32
In summary, the findings presented here expand the documented constellation of diiron enzyme 33
catalysis to include a dioxygenase reactivity in which an unactivated alkene is converted to a vicinal diol. 34
35
36
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INTRODUCTION 37
Diiron clusters within the active sites of enzymes facilitate the binding of molecular oxygen and its 38
derivatives and are able to perform redox chemistry, which results in a range of chemical outcomes 39
(Edmondson and Juynh, 1996). All diiron enzymes characterized to date belong to one of two separate 40
classes, one soluble and the other membrane bound (Shanklin and Somerville, 1991). Both classes have 41
the ability to catalyze the oxidation of unactivated C-H bonds to give a range of chemical outcomes 42
(Shanklin and Cahoon, 1998; Fox et al., 2004). For instance, both soluble and membrane diiron enzyme 43
classes contain desaturase enzymes that perform the stereo- and regioselective introduction of Z- (cis) 44
double bonds into unactivated lipid acyl chains. The reactions are thought to proceed via a radical 45
mechanism initiated by abstraction of a specific hydrogen from substrate (Buist, 2004). Double bond 46
formation ensues via the abstraction of a second neighboring hydrogen. As predicted by Bloch (Bloch, 47
1969) and subsequently confirmed by X-ray crystallography (Lindqvist et al., 1996; Bai et al., 2015), the 48
boomerang shape of the substrate binding channel within the desaturase drives the formation of the 49
(Z)-olefinic fatty acids. 50
There is a diverse constellation of chemical outcomes performed by variant enzymes that are 51
structurally related to the prototypical desaturase. The membrane-bound diiron-containing plant fatty 52
acid desaturase (FAD) family of FAD2 variant enzymes perform a variety of chemical transformations. 53
Using oleate as substrate, either desaturated or hydroxylated products are obtained; using linoleate as a 54
substrate, the corresponding epoxide, a conjugated double bond, or an acetylenic bond can be 55
produced. Changes in chemoselectivity are based on a relatively small number of amino acid sequence 56
differences which presumably alter the relative orientation of the substrate with respect to the active 57
site oxidant (Bhar et al., 2012). For instance, changes to only four amino acid side chains was sufficient 58
to predominantly convert a FAD2 into a hydroxylase and vice versa (Broun et al., 1998; Broadwater et 59
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al., 2002). Despite our increasing understanding of specificity determining residues within the FAD2-60
related diiron enzymes, further interpretation has been hindered by the lack of structural information 61
for these enzymes. Recently published structures of several mammalian membrane-bound desaturases 62
suggest it will be possible to solve one of the plant FAD2 class at some point and we will be able to 63
correlate changes to the enzyme structure with distinct functional outcomes (Bai et al., 2015; Wang et 64
al., 2015). 65
The soluble class of desaturase enzymes exemplified by the castor (Ricinus communis) 918:0-ACP 66
desaturase (Lindqvist, 2001) has been shown to contain members that display a variety of chain-length 67
specificities and regioselectivities (Shanklin et al., 2009). Mechanisms have been proposed for both 68
chain length specificity (Cahoon et al., 1997; Whittle and Shanklin, 2001) and for regioselectivity (Guy et 69
al., 2011). During our studies on regioselectivity, we engineered a triple mutant of the castor acyl-ACP 70
desaturase (T117R/G188L/D280K) that converts stearoyl-ACP into an allylic alcohol trans-isomer (E)-10-71
18:1-9-OH via a (Z)-9-18:1 intermediate (Whittle et al., 2008). This work described a soluble desaturase 72
acting as an olefin oxygenase similar in behavior to that displayed by another soluble diiron protein, 73
methane monooxygenase (Gherman et al., 2004). We showed that the conversion of (Z)-9-18:1 74
substrate to (E)-10-18:1-9-OH product by castor desaturase T117R/G188L/D280K proceeds via hydrogen 75
abstraction at C-11 and highly regioselective hydroxylation (>97%) at C-9 (Whittle et al., 2008). 18O-76
labeling studies show that the hydroxyl oxygen in the reaction product is exclusively derived from 77
molecular oxygen. 78
The present work was initially designed to evaluate the individual contributions of T117R, G188L, and 79
D280K in castor desaturase to allylic alcohol formation. During these experiments, we discovered a 80
novel dioxygenase reactivity of the soluble desaturase that results in the conversion of oleoyl-ACP to 81
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erythro-9, 10-dihydroxystearate. The same product was found in TMS-derivatized methyl esters from 82
castor seed where it constitutes approximately 0.7% of the total fatty acids 83
84
RESULTS 85
As part of our continuing structure-function analysis of diiron enzymes, we analyzed the contributions of 86
each of the mutations within the castor desaturase T117R/G188L/D280K triple mutant that converts 87
oleoyl-ACP into (E)-10-18:1-9-OH (Whittle et al., 2008). Each of the individual mutants was constructed 88
and tested for its activity using oleoyl-ACP as substrate. In each case, the product profiles were 89
determined by GC-MS analysis. The results are shown in Figure 1. The GC elution profile of substrate is 90
shown in Panel A (Fig. 1) and features a peak corresponding to 18:19 methyl ester (peak 1). A minor 91
shoulder peak can be attributed to 18:111 (peak 2) and is a well-known artefact of the expression 92
system. As shown in Panel B (Fig. 1), the triple mutant T117R/G188L/D280K converted most of the 93
oleoyl-ACP substrate into a mixture of the Z(cis)18:1Δ10 9OH (peak 3) and E(trans) 18:1Δ10 9OH allylic 94
alcohol (peak 4) isomers, with the E form predominating by approximately 3-fold over the Z form. 95
Reactivity of the Castor Desaturase Single Mutants T117R, G188L, and D280K. 96
Each of the single mutants was active with respect to the oleoyl-ACP substrate (Fig. 1, C, D and E). The 97
T117R mutant produced approximately 15-fold more of the E 18:1Δ10 9OH isomer than the 98
corresponding Z isomer. However, a new peak (labeled 5 in Fig. 1C) became apparent at an elution time 99
that was not characteristic of the silylated derivatives of commonly occurring fatty acid methyl esters. 100
The G188L mutant produced approximately a 1:1 mixture of E and Z isomers of 18:1Δ10 9OH (Fig. 1D), 101
but no detectable trace of the novel fatty acid species (peak 5) was produced by the T117R mutant. The 102
D280K mutant was less active than T117R and G188L, producing only a small amount of the E isomer of 103
18:1Δ10 9OH (Fig. 1E), along with a small amount of the novel fatty acid (peak 5). As expected, the wild-104
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type desaturase showed very little activity with its natural product oleoyl-ACP, but close inspection 105
revealed the production of a trace of novel species (peak 5) based on its elution time and mass spectra 106
(Fig. 1F). 107
The novel fatty acid product (peak 5) is 9,10-dihydroxystearate. 108
Mass-spectral analysis of the peak-5 product produced by the T117R mutant (Fig. 1C) revealed a 109
molecular ion of 474 AMU, consistent with an 18C fatty acid methyl ester containing two silylated 110
hydroxyl groups (Fig. 2A). Fragmentation of the product between the two silyl groups produced 111
fragments of 259 AMU for the carboxyl-containing fragment and 215 AMU for the methyl-containing 112
fragment (diagrammed in Fig. 2B), consistent with the presence of vicinal hydroxyl groups at C9 and C10. 113
The identity of the peak-5 product was confirmed by comparison of its fragmentation pattern with that 114
of a silylated authentic commercial standard of erythro-methyl 9,10-dihydroxy stearate (Fig. 2C). 115
Analysis of the peak-5 product from the D280K mutant also showed the same fragmentation pattern. 116
9,10-Dihydroxystearate produced by the T117R mutant is solely in the erythro configuration. 117
Fatty acids containing vicinal mid-chain hydroxy groups may exist as threo or erythro diastereoisomers 118
(Fig. 3). To distinguish between these possibilities, we compared the GC elution times of the novel 119
product from T117R with those of authentic threo and erythro-9, 10-dihydroxystearate standards (Fig. 4, 120
A, B, and C, respectively). The T117R product eluted as a single defined peak without any detectable 121
shoulders (Fig. 3A) and coeluted with authentic erythro standard (Fig. 4C). The authentic threo standard 122
(Fig. 4B) eluted ahead of that of the T117R product (Fig. 4A). When a small amount of the T117R product 123
was mixed with either the threo standard (Fig. 4D) or the erythro standard (Fig. 4E), two peaks were 124
seen for the sample spiked with threo standard whereas a single coeluting peak was seen for the sample 125
spiked with erythro standard. These results confirm the assignment of the T117R product as erythro-9, 126
10-dihydroxystearate. 127
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The hydroxyl oxygens at both C9 and C10 are derived from molecular oxygen. 128
The oxygen atoms in either of the two hydroxyl groups could in principle arise from water or molecular 129
oxygen (Fig. 5). To distinguish between these possibilities, T117R, oleoyl-ACP, and all assay components 130
were first degassed by multiple gas exchange cycles employing vacuum and O2-free argon with the use 131
of a Schlenk line (Arnold and Bohle, 1996) to remove residual atmospheric 16O2 from the sealed reaction 132
vials. Assay reactions were subsequently incubated in the presence of 16O2 or 18O2. We used mass-133
labeled 18:1 d2-11,11 oleoyl-ACP for these assays to ensure the product we observed was derived from 134
the enzymatic reaction rather than from endogenous oleate contaminant. Analysis of the methylated 135
silylated products from reaction under air yielded the expected 217 and 259 AMU products (the methyl 136
fragment increased by 2 AMU relative to unlabeled product results from the substitution for the two 137
hydrogens at C11 for deuterons (Fig. 6A)). The same experiment performed under 18O2 resulted in the 138
production of fragments of 219 and 261 AMU, consistent with the incorporation of one 18O at each of 139
the hydroxyl positions. 140
The formation of 9, 10-dihyroxystearate from oleate is the result of a dioxygenase reaction. 141
The incorporation of molecular oxygen at the 9 and 10 positions of oleate could in principle result from a 142
single dioxygenase reaction, or from two sequential monooxygenase reactions. To distinguish between 143
these possibilities, we degassed the samples as described above and performed a reaction under an 144
atmosphere containing an equimolar fraction of 16O2 and 18O2 (Fig. 7B) and performed mass 145
spectrometry on methylated acetonide derivatives of the product (Fig 7E). Acetonide derivatives were 146
used because they protect vicinal hydroxy groups while maximizing the detectable mass ion of the 147
product. If the reaction operates via a dioxygenase mechanism, then the oxygen atoms at both hydroxyl 148
positions should derive exclusively from either 16O2 or 18O2, resulting in either M or M+4 species. 149
Alternatively, if the mechanism employs two sequential monooxygenase reactions, a 1:2:1 pattern of 150
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M:M+2:M+4 would be expected by random incorporation of either 16O or 18O at each hydroxyl position. 151
Consistent with a dioxygenase mechanism, reactions performed under an equimolar mix of 16O2 and 18O2 152
yielded only M and M+4 peaks (355 and 359), with no detectable 357 species (Fig. 7B). Individual control 153
16O2 and 18O2 reactions only showed the expected 355 and 359 major species accompanied by minor 154
peaks at M+1 and M+2 that approximate the natural abundance of 13C (Fig. 7, A and C, respectively). 155
That M+1 and M+2 peaks originate from natural 13C was confirmed by the fragmentation of equivalent 156
derivatives of an authentic erythro-9, 10-dihydroxystearate, which showed the same proportions of M, 157
M+1, and M+2 species (Fig. 7D). 158
The native castor desaturase can convert oleoyl-ACP to 9,10-dihydroxystearate. 159
The formation of dihydroxystearate with selected mutated desaturases prompted us to probe for the 160
formation of this compound by the wild-type enzyme. Interestingly, using a prolonged time of 161
incubation (240 min) with oleoyl-ACP as substrate, we were able to identify production of 9, 10-162
dihydroxystearate (peak 5) at low levels (Fig. 8). This compound was accompanied by lesser amounts of 163
E 18:110 9 OH (peak 4). 164
Castor oil contains erythro-9,10-dihydroxystearate. 165
The observation that the native castor desaturase can produce small amounts of 9,10-dihydroxystearate 166
correlates well with an early report by King et al (King, 1942) who isolated a small amount of 9,10-167
dihydroxystearate from castor oil. We sought to confirm this observation and analyzed a fatty acid 168
extract of castor seeds by GC-MS after methylation and silylation. Chromatograms of castor seed fatty 169
acid derivatives (Fig. 9A) showed the expected common C16 and C18 fatty acids, along with a major 170
peak of ricinoleic acid which is followed by a small discrete peak (labeled 8 in Fig. 9A inset) of 171
approximately 0.7% (of total fatty acids), which corresponds to the elution time of disilylated methyl 9, 172
10-dihydroxystearate. Mass spectral analysis of this peak revealed fragments of 215 and 259 AMU 173
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confirming its assignment as 9, 10-dihydroxystearate (compare Fig. 9B with Fig. 2A and C). Based on the 174
in vitro assays using purified enzyme reported above, we hypothesize that this 9,10 dihydroxystearate 175
arises from the dioxygenation of oleoyl-ACP product of the stearoyl-ACP desaturase. If this were the 176
case, the 9,10-dihydroxystearate would be in the erythro form as originally proposed (Morris and 177
Crouchman, 1972). We therefore conducted coelution studies with authentic threo or erythro standards 178
(Fig. 9, C-E). The 9, 10 dihydroxystearate isolated from castor eluted as a single peak (Fig. 9C) with the 179
same mobility as that of the authentic erythro standard (Fig. 9E). By contrast, two peaks were seen in 180
the spiking experiment using threo standard (Fig. 9D). 181
182
183
DISCUSSION 184
Stereoselective dihydroxylation reactions are important to the chemical industry (Borrell and Costas, 185
2017) since diols serve as valuable synthons. The osmium-based asymmetric dihydroxylation reaction 186
(Crispino and Sharpless, 1993) is a prominent example of controlled olefin oxidation and was (in part) 187
recognized by the award of the 2001 Nobel Prize in Chemistry to its inventor, K. B. Sharpless. In addition, 188
biocatalytic diol formation from aromatics by whole-cell mutant Pseudomonas cultures has furnished 189
the synthetic chemist with a variety of enantiomerically pure cyclohexadiene-cis-diols. (Hudlicky and 190
Thorpe, 1996). Much effort has also been expended to develop iron-based biomimetic catalytic 191
methodology for this reaction (Oloo and Que, 2015). Herein, we report the details of our investigation 192
into a “green chemical approach”: the castor 918:0-ACP desaturase-mediated syn-dihydroxylation of an 193
unactivated alkene in the form of oleoyl-ACP to erythro -9,10-dihydroxystearoyl-ACP. 194
Stearoyl-ACP desaturase belongs to the non-heme diiron subclass of oxidative enzymes that have been 195
shown to mediate a variety of chemical transformations including dehydrogenation and 196
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monoxygenation. Typical products include primary, secondary, and allylic alcohols in addition to the 197
conversion of double bonds to epoxides (Wallar and Lipscomb, 1996). However, a diiron center 198
performing dioxygen chemistry to convert a double bond to a vicinal diol as reported here is without 199
precedent. The closest comparable example we are aware of is arylamine oxygenase (CmlI) from the 200
chloramphenicol biosynthesis pathway, which incorporates two oxygens from O2 into the aryl‐nitro 201
product; however, this occurs in two consecutive monooxygenations (Komor et al., 2017). We envision 202
the conversion of alkene to vicinal erythro-diol in this work to be mechanistically related (Fig. 4) to that 203
described for Rieske cis-diol-forming dioxygenases (Ensley et al., 1982; Karlsson et al., 2003). More 204
specifically, we envision involvement of a bridged hydroperoxo-diiron species similar to that proposed 205
by Solomon and Srnec (Chalupsky et al., 2014) for the conversion of stearate to oleate by two 206
consecutive hydrogen atom abstractions: “ - CH2-CH2-“ to “-CH=CH-“ . When presented with an alkene 207
moiety, the vinyl hydrogens are unavailable for abstraction for steric reasons and this same species is 208
forced to transfer two oxygen atoms to substrate as shown in Fig. 4 (Pathway 1). Our oxygen-labelling 209
experiments rule out an epoxidation/hydrolysis route (Pathway 2). It is possible that our T117R mutant 210
may change the molecular architecture of the substrate binding cavity, altering the relative orientation 211
of the substrate with respect to the hydroperoxo-diiron group and facilitating deoxygenation relative to 212
the wild-type enzyme. That the diol is produced as the erythro diastereoisomer, in which both hydroxy 213
groups occur on one face (Fig. 3), is consistent with the geometry of the active site substrate-binding 214
cavity with respect to the diiron active site oxidant (Lindqvist et al., 1996), in which stearate binds in a 215
quasi-eclipsed conformation at C9 and C10, projecting the pro-(R) hydrogens towards the active site 216
oxidant (Behrouzian et al., 2002). Future availability of a crystal structure of the T117R mutant in 217
complex with bound oleoyl-ACP, or of the T117R mutant alone or with substrate bound as previously 218
modeled (Whittle et al., 2008), would be useful starting points for probing mechanistic models using 219
computational methods such as density functional theory. Indeed, homology modeling was recently 220
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shown to be a useful approach for elucidating selectivity mechanisms of desaturase enzymes such as 221
FAD2 and FAD3 (Cai et al., 2018). 222
223
The low levels of 9, 10-dihydroxystearate in castor suggests that this system is not optimized to produce 224
this particular product. Higher levels of the diol may accumulate via enzymes with active site geometries 225
that permit more efficient dioxygenation. Cardimine impatiens is an example of a plant that accumulates 226
approximately 25% of 9, 10-dihydroxystearate (and its chain-elongation products) in its seed oil 227
(Mikolajczak et al., 1964). It is tempting to speculate that it contains a desaturase that has undergone 228
mutation/selection to optimize the production of the diol from the initial alkene product. Examples of 229
desaturases with multiple sequential oxidation activity include English ivy (Hedera helix) which can 230
perform 9- followed by 4 desaturation on stearoyl-ACP (Guy et al., 2007); FM1, a fungal membrane 231
desaturases that sequentially inserts a 12 followed by a 15 double bond into oleoyl-phosphatidyl 232
ethanolamine (Cai et al., 2018); and an insect multifunctional enzyme that functions as a 11 233
desaturase, 11 acetylenase, and 13 desaturase (Serra et al., 2007). 234
Major oxygenated fatty acids such as ricinoleic- and vernolic acids are typically produced in the 235
endoplasmic reticulum by variant FAD2 membrane-bound desaturases (van de Loo et al., 1995; Lee et 236
al., 1998). On the other hand, fatty acids with unusual double bond positions such as 16:14, 16:19, 237
and 18:1 6 are synthesized within the plastid (Shanklin and Cahoon, 1998). Thus, the production of 238
oxygenated fatty acids such as the erythro-9, 10-dihydroxystearate in the plastid as reported here is very 239
unusual if not unique. It is likely that in species with high levels of accumulation such as C. impatiens, 240
there exists a variant acyl-ACP thioesterase that cleaves the vicinal diol fatty acid from its ACP adduct in 241
addition to specialized acyltransferases and other components that facilitate its transfer from the plastid 242
to triglyceride storage lipids. 243
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More than 70 years ago, 9,10-dihydroxystearate was reported as a component of castor oil (King, 1942) 244
at approximately 1% of the total fatty acids (Sreenivasan et al., 1956). The stereochemistry of the diol 245
was later determined to be the erythro configuration (Morris and Crouchman, 1972). Consistent with 246
these earlier reports, castor oil samples evaluated in the present work contained approximately 0.7% of 247
erythro -9,10-dihydroxystearate. That the wild-type castor desaturase can produce this compound was 248
an entirely unanticipated result and resolves a long-standing mystery. In addition, our results 249
underscore the remarkable plasticity of the non-heme diiron catalytic center found in the desaturase 250
family of enzymes. It appears that subtle changes in the active site architecture found in these versatile 251
oxidants can allow new reaction pathways to emerge. Further detailed mechanistic work is needed to 252
understand the relationship between reaction outcome and details of the active site architecture. 253
MATERIALS AND METHODS 254
Mutant construction 255
Synthesis of the castor (Ricinus communis) desaturase triple mutant T117R/G188L/D280K and D280K 256
single mutants were previously described (Whittle et al., 2008; Guy et al., 2011). The single mutants 257
T117R and G188L were identified by mutagenesis-selection experiments (Whittle and Shanklin, 2001). 258
The open reading frames were introduced into pET9d using XbaI and EcoRI restriction sites and the 259
resulting clones were validated by sequencing. 260
Mutant Analysis 261
Desaturases, and variants thereof, were overexpressed in E. coli BL21(DE3) with the use of pET9d. 262
Recombinant desaturase was enriched to >90% purity by 20CM cation exchange chromatography 263
(Applied Biosystems). Desaturation reactions (600 µl) (Cahoon and Shanklin, 2000) were performed by 264
incubation of the desaturase with 18:0- and 18:1-ACP substrates in the presence of recombinant spinach 265
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ACP-I (Beremand et al., 1987). Uniformly deuterated stearate was obtained from Cambridge Isotope 266
Laboratories, Andover MA, and 9,10 d2 oleate and 11, 11 d2 oleate was obtained from the collection of 267
Tulloch (Tulloch, 1983). Experiments reported herein were replicated three or more times and 268
representative results are presented. 269
Fatty acid analysis 270
Fatty acid methyl esters (FAMEs) were prepared by addition of 2 ml of 1% NaOCH3 v/v in methanol and 271
incubated for 60 min at 50°C. Fatty acid methyl esters were extracted twice into 2 ml hexane after 272
acidification with 100 µl of glacial acetic acid. Hexane was evaporated to dryness under a stream of N2, 273
and samples were resuspended in hexane for GC analysis. FAMEs were dried and resuspended in 100 µl 274
of BSTFA (N,O-bis(trimethylsilyl) trifluoroacetamide) and TMCS (trimethylchlorosilane) (Supelco) for 45 275
min at 60°C to create trimethyl silyl derivatives. Samples were analyzed with an HP5890 gas 276
chromatograph (Agilent) fitted with a 60 m x 250 µm SP-2340 capillary column (Supelco). The oven 277
temperature was raised from 100°C to 160°C at a rate of 25°C min–1 and from 160°C to 240°C at a rate of 278
10°C min–1 with a flow rate of 1.1 ml min–1. Mass spectra were analyzed using an HP5973 mass selective 279
detector (Agilent). For 18O experiments, oxygen was removed from the sample cell by repeated 280
evacuation and purging of the cell with O2-free argon using a Schlenk line. Two mixtures were prepared, 281
one containing desaturase enzyme, buffer, ferredoxin NADPH(+) reductase, and substrate, the other 282
containing ferredoxin and NADPH. The two anaerobic mixtures were transferred to sealed reaction vials 283
containing an atmosphere composed of either 16O2, 18O2 (Cambridge Isotope Laboratories, Andover MA), 284
or an equimolar mixture of 16O2 and 18O2. Reactions were terminated by the addition of toluene, and 285
fatty acids were esterified and silylated as described above for experiments designed to fragment the 286
fatty acid to reveal the position of the vicinal hydroxyl groups. Alternatively, for the labelled oxygen 287
experiments designed to determine the reaction mechanism, fatty acids were converted to methyl 288
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esters after which vicinal hydroxy groups were converted to their acetonide derivatives (Singh et al., 289
2008). To achieve this, methyl ester samples were dried under nitrogen and resuspended in 40 µl of 4 290
mM ZrCl4 catalyst in diethyl ether, 200 µl dichloromethane (CH2Cl2), and 5 ul dimethoxypropane. The 291
mixture was incubated with shaking at 22°C for 2 hrs. The mixture was extracted with 3 ml chloroform 292
(CHCl3) and 1 ml water, separated by centrifugation (at 1,500g for 5 min.) and the lower phase was 293
collected and dried under nitrogen before resuspension in hexane for GC/MS analysis. Samples 294
were analyzed on an HP6890/5973 GC/MS equipped with a 30 m x 250 µm HP 5MS capillary 295
column (Supelco). Oven temperature was held at 100°C for 2 min, raised to 300°C at the rate of 20°C 296
min-1, and held for 2 min. 297
298
Accession Numbers 299
Sequence data from this article can be found in the GenBank/EMBL data libraries under accession 300
number M59857. 301
Acknowledgements. 302
This work was funded by the U.S. Department of Energy, Office of Science, Office of Basic Energy 303
Sciences under contract number DE-SC0012704 to J.S. We thank Dr. John Lipscomb, Dr. Diane Cabelli 304
and Dr. Xio-Hong Yu for helpful discussion and Dr. Pat Covello for providing some of the deuterated 305
compounds prepared by Tulloch. 306
307
Figure Legends 308
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Fig. 1 GC-MS elution profiles of TMS derivatives. Chromatograms of TMS derivatives of 18:1-ACP 309
substrate (A) and product distributions for the castor desaturase triple mutant T117R G188L D280K (B), 310
and each of the single mutants T117R (C), G188L (D), and D280K (E) reveals a novel fatty acid species 311
labeled as peak 5. Product profile of wild-type castor desaturase is included (F) as a control. Peak 312
identities: Z18:1Δ9, (1); Z18:1Δ11, (2); Z18:1Δ10 9OH, (3); E18:1Δ10 9OH, (4). 313
Fig. 2. The novel fatty acid product is 9, 10-dihydroxystearate. Comparison of mass spectra of TMS 314
derivatives of the novel enzymatic product produced by the castor desaturase T117R mutant (A) and an 315
authentic erythro 9,10 dihydroxy stearate standard (C), and the fragmentation pattern giving rise to the 316
major ions at 215 and 259 AMU (B). 317
Fig. 3. The structural relationships of compounds discussed in this work. 1 Stearoyl ACP, showing two 318
hydrogens at C-9, 10 that are removed by desaturase; 2 Oleoyl ACP, the product of stearoyl 9,10 319
desaturation; 3 Erythro-9(R) ,10 (R)-dihydroxystearoyl ACP, the predicted product of 1 step direct oleate 320
dihydroxylation; 4 Threo-9(S) ,10 (R)-dihydroxystearoyl ACP, a possible product of enzymatic 2 step 321
oleate epoxidation/hydrolysis sequence. 322
Fig. 4. The 9,10-dihydroxystearate produced by the castor T117R mutant is solely in the erythro 323
configuration. Gas chromatograms of 9,10-dihydroxy- stearates are compared for the reaction product 324
of T117R (A) to those of standards: threo configuration (B), the erythro configuration (C), a mixture of 325
the T117R product and the threo standard (D), and the T117R product and the erythro standard (E). 326
Fig. 5. Two potential schemes for the conversion of oleate to erythro 9,10 dihydroxystearate by a 327
diiron-containing desaturase-dioxygenase. The initial bridged hydroperoxo species in both mechanisms 328
is inspired by large-scale multireference ab initio calculations on a related enzyme (Chalupsky et al, 329
2014). 330
www.plantphysiol.orgon October 31, 2020 - Published by Downloaded from Copyright © 2019 American Society of Plant Biologists. All rights reserved.
16
Fig. 6. Both hydroxyl oxygens of 9,10-dihydroxystearate are derived from molecular oxygen. TMS 331
derivatives of 9, 10-dihydroxystearate product from the castor desaturase T117R mutant using 18:1 11-332
d2- substrate under air (A) or 18O2 shown in the diagram as O* (B). 333
Fig. 7. 9,10-dihydroxy stearate formation is the result of a single dioxygenase reaction. 334
Chromatograms and corresponding mass spectra of acetonide derivatives of 9,10 dihydroxy stearate 335
from reactions carried out under 16O2 (A), equimolar 16O2 and 18O2 (B), and 18O2 (C). Also depicted is an 336
authentic erythro 9,10 dihydroxy stearate standard (D) along with a diagram of its fragmentation (E). 337
Fig. 8. Upon prolonged incubation, the castor wild-type desaturase can convert 18:1 substrate to 338
erythro-9, 10-dihydroxystearate. Peak identities: Z18:1Δ9, (1); Z18:1Δ11, (2); Z18:1Δ10 9OH, (3); 339
E18:1Δ10 9OH; (4), and 9,10-dihydroxystearate (5). 340
Fig. 9. Low-level erythro-9, 10-dihydroxystearate is present in developing castor embryos. Gas 341
chromatogram of TMS derivatives of castor embryos (A) and the mass spectrum corresponding to peak 342
8, i.e., 9,10-dihydroxystearate (B). Peak identities: 16:0 (1), 18:0 (2), 18:1D9 (3), 18:1D11 (4), 18:2D9,12 343
(5), 12-OH 18:1D9 (6), 18:3D9,12,15 (7), and 9, 10 OH 18:0 (8). C-E, GC peaks for TMS derivatives of 9,10 344
dihydroxystearate from castor embryo (C), 9,10-dihydroxystearate from castor developing embryos 345
mixed with authentic threo-9,10-dihydroxystearate standard (D), and authentic erythro-9,10-346
dihydroxystearate standard (E). 347
348
REFERENCES 349
Arnold EV, Bohle DS (1996) Isolation and oxygenation reactions of nitrosylmyoglobins. Nitric Oxide, Pt B 350 269: 41-55 351
Bai Y, McCoy JG, Levin EJ, Sobrado P, Rajashankar KR, Fox BG, Zhou M (2015) X-ray structure of a 352 mammalian stearoyl-CoA desaturase. Nature 524: 252-256 353
www.plantphysiol.orgon October 31, 2020 - Published by Downloaded from Copyright © 2019 American Society of Plant Biologists. All rights reserved.
17
Behrouzian B, Savile CK, Dawson B, Buist PH, Shanklin J (2002) Exploring the hydroxylation-354 dehydrogenation connection: novel catalytic activity of castor stearoyl-ACP Delta(9) desaturase. 355 J Am Chem Soc 124: 3277-3283. 356
Beremand PD, Hannapel DJ, Guerra DJ, Kuhn DN, Ohlrogge JB (1987) Synthesis, cloning, and expression 357 in Escherichia coli of a spinach acyl carrier protein-I gene. Arch. Biochem. Biophys. 256: 90-100 358
Bhar P, Reed DW, Covello PS, Buist PH (2012) Topological Study of Mechanistic Diversity in Conjugated 359 Fatty Acid Biosynthesis. Angewandte Chemie-International Edition 51: 6686-6690 360
Bloch K (1969) Enzymatic synthesis of monounsaturated fatty acids. Acc. Chem. Res. 2: 193-202 361 Borrell M, Costas M (2017) Mechanistically Driven Development of an Iron Catalyst for Selective Syn-362
Dihydroxylation of Alkenes with Aqueous Hydrogen Peroxide. Journal of the American Chemical 363 Society 139: 12821-12829 364
Broadwater JA, Whittle E, Shanklin J (2002) Desaturation and hydroxylation. Residues 148 and 324 of 365 Arabidopsis FAD2, in addition to substrate chain length, exert a major influence in partitioning of 366 catalytic specificity. J Biol Chem 277: 15613-15620 367
Broun P, Shanklin J, Whittle E, Somerville C (1998) Catalytic plasticity of fatty acid modification enzymes 368 underlying chemical diversity of plant lipids. Science 282: 1315-1317 369
Buist PH (2004) Fatty acid desaturases: selecting the dehydrogenation channel. Nat Prod Rep 21: 249-370 262. 371
Cahoon EB, Lindqvist Y, Schneider G, Shanklin J (1997) Redesign of soluble fatty acid desaturases from 372 plants for altered substrate specificity and double bond position. Proc. Natl. Acad. Sci. USA 94: 373 4872-4877 374
Cahoon EB, Shanklin J (2000) Substrate-dependent mutant complementation to select fatty acid 375 desaturase variants for metabolic engineering of plant seed oils. Proc Natl Acad Sci U S A 97: 376 12350-12355 377
Cai YH, Yu XH, Liu Q, Liu CJ, Shanklin J (2018) Two clusters of residues contribute to the activity and 378 substrate specificity of Fm1, a bifunctional oleate and linoleate desaturase of fungal origin. 379 Journal of Biological Chemistry 293: 19844-19853 380
Chalupsky J, Rokob TA, Kurashige Y, Yanai T, Soomon EI, Rulisek L, Srnec M (2014) Reactivity of the 381 Binuclear Non-Heme Iron Active Site of Delta(9) Desaturase Studied by Large-Scale 382 Multireference Ab Initio Calculations. Journal of the American Chemical Society 136: 15977-383 15991 384
Crispino GA, Sharpless KB (1993) Enantioselective Synthesis of Juvenile Hormone-Iii in 3 Steps from 385 Methyl Farnesoate. Synthesis-Stuttgart: 777-779 386
Edmondson DE, Juynh BH (1996) Diiron cluster intermediates in biological oxygen activation reactions. 387 Inorg. Chimica Acta 252: 399-404 388
Ensley BD, Gibson DT, Laborde AL (1982) Oxidation of Naphthalene by a Multicomponent Enzyme-389 System from Pseudomonas Sp Strain Ncib9816. Journal of Bacteriology 149: 948-954 390
Fox BG, Lyle KS, Rogge CE (2004) Reactions of the diiron enzyme stearoyl-acyl carrier protein 391 desaturase. Acc Chem Res 37: 421-429 392
Gherman BF, Baik MH, Lippard SJ, Friesner RA (2004) Dioxygen activation in methane monooxygenase: 393 A theoretical study. Journal of the American Chemical Society 126: 2978-2990 394
Guy JE, Whittle E, Kumaran D, Lindqvist Y, Shanklin J (2007) The crystal structure of the ivy Delta4-16:0-395 ACP desaturase reveals structural details of the oxidized active site and potential determinants of 396 regioselectivity. J Biol Chem 282: 397
: 19863-19871 398
www.plantphysiol.orgon October 31, 2020 - Published by Downloaded from Copyright © 2019 American Society of Plant Biologists. All rights reserved.
18
Guy JE, Whittle E, Moche M, Lengqvist J, Lindqvist Y, Shanklin J (2011) Remote control of 399 regioselectivity in acyl-acyl carrier protein-desaturases. Proc Natl Acad Sci U S A 108: 16594-400 16599 401
Hudlicky T, Thorpe AJ (1996) Current status and future perspectives of cyclohexadiene-cis-diols in 402 organic synthesis: Versatile intermediates in the concise design of natural products. Chemical 403 Communications: 1993-2000 404
Karlsson A, Parales JV, Parales RE, Gibson DT, Eklund H, Ramaswamy S (2003) Crystal structure of 405 naphthalene dioxygenase: Side-on binding of dioxygen to iron. Science 299: 1039-1042 406
King G (1942) The dihydroxystearitc acid of castor oil - Its constitution and structural relationship to the 407 9 10-dihydroxystearic acids m p's 132 degrees and 95 degrees. Journal of the Chemical Society: 408 387-391 409
Komor AJ, Rivard BS, Fan RX, Guo YS, Que L, Lipscomb JD (2017) CmII N-Oxygenase Catalyzes the Final 410 Three Steps in Chloramphenicol Biosynthesis without Dissociation of Intermediates. 411 Biochemistry 56: 4940-4950 412
Lee M, Lenman M, Banas A, Bafor M, Singh S, Schweizer M, Nilsson R, Liljenberg C, Dahlqvist A, 413 Gummeson PO, Sjodahl S, Green A, Stymne S (1998) Identification of non-heme diiron proteins 414 that catalyze triple bond and epoxy group formation. Science 280: 915-918 415
Lindqvist Y (2001) Delta nine stearoyl-acyl carrier protein desaturase. John Wiley & Sons, Chichester UK 416 Lindqvist Y, Huang W, Schneider G, Shanklin J (1996) Crystal structure of a delta nine stearoyl-acyl 417
carrier protein desaturase from castor seed and its relationship to other diiron proteins. EMBO J. 418 15: 4081-4092 419
Mikolajczak KL, Wolff IA, Smith CR (1964) Dihydroxy Fatty Acids in Petroleum Ether Extract of 420 Cardamine Impatiens Seed. Journal of the American Oil Chemists Society 41: 22-& 421
Morris LJ, Crouchman ML (1972) Absolute Optical Configurations of Isomeric 9,10-Epoxystearic, 9,10-422 Dihydroxystearic and 9,10,12-Trihydroxystearic Acids. Lipids 7: 372-+ 423
Oloo WN, Que L (2015) Bioinspired Nonheme Iron Catalysts for C-H and C=C Bond Oxidation: Insights 424 into the Nature of the Metal-Based Oxidants. Accounts of Chemical Research 48: 2612-2621 425
Serra M, Pina B, Abad JL, Camps F, Fabrias G (2007) A multifunctional desaturase involved in the 426 biosynthesis of the processionary moth sex pheromone. Proc Natl Acad Sci U S A 104: 16444-427 16449 428
Shanklin J, Cahoon EB (1998) Desaturation and related modifications of fatty acids. Annu. Rev. Plant 429 Physiol. Plant. Mol. biol. 49: 611-641 430
Shanklin J, Guy JE, Mishra G, Lindqvist Y (2009) Desaturases - emerging models for understanding 431 functional diversification of diiron-containing enzymes. J Biol Chem 284 18559- 18563 432
Shanklin J, Somerville C (1991) Stearoyl-acyl-carrier-protein desaturase from higher plants is structurally 433 unrelated to the animal and fungal homologs. Proc. Natl. Acad. Sci. USA 88: 2510-2514 434
Singh S, Duffy CD, Shah STA, Guiry PJ (2008) ZrCl(4) as an efficient catalyst for a novel one-pot 435 protection/deprotection synthetic methodology. Journal of Organic Chemistry 73: 6429-6432 436
Sreenivasan B, Kamath NR, Kane JG (1956) Studies on Castor Oil .1. Fatty Acid Composition of Castor 437 Oil. Journal of the American Oil Chemists Society 33: 61-66 438
Tulloch AP (1983) Synthesis, analysis and application of specifically deuterated lipids. Prog Lipid Res 22: 439 235-256 440
van de Loo FJ, Broun P, Turner S, Somerville C (1995) An oleate 12-hydroxylase from Ricinus communis 441 L. is a fatty acyl desaturase homolog. Proc. Natl. Acad. Sci. USA 92: 6743-6747 442
Wallar BJ, Lipscomb JD (1996) Dioxygen activation by enzymes containing binuclear non-heme iron 443 clusters. Chem. Rev. 96: 2625-2657 444
Wang H, Klein MG, Zou H, Lane W, Snell G, Levin I, Li K, Sang BC (2015) Crystal structure of human 445 stearoyl-coenzyme A desaturase in complex with substrate. Nat Struct Mol Biol 22: 581-585 446
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19
Whittle E, Shanklin J (2001) Engineering delta 9-16:0-acyl carrier protein (ACP) desaturase specificity 447 based on combinatorial saturation mutagenesis and logical redesign of the castor delta 9-18:0-448 ACP desaturase. J Biol Chem 276: 21500-21505 449
Whittle EJ, Tremblay AE, Buist PH, Shanklin J (2008) Revealing the catalytic potential of an acyl-ACP 450 desaturase: tandem selective oxidation of saturated fatty acids. Proc Natl Acad Sci U S A 105: 451 14738-14743 452
www.plantphysiol.orgon October 31, 2020 - Published by Downloaded from Copyright © 2019 American Society of Plant Biologists. All rights reserved.
4A B C
ED
Det
ecto
r R
esp
on
seD
etec
tor
Res
po
nse
Retention Time (min.)
Retention Time (min.)
1
2
1
2 3
5
1 23
4
1
2
45
12
3 4
F
1
25
9.0 9.4 9.8 10.29.0 9.4 9.8 10.29.0 9.4 9.8 10.2
9.0 9.4 9.8 10.29.0 9.4 9.8 10.29.0 9.4 9.8 10.2
Fig. 1 GC-MS elution profiles of TMS derivatives. Chromatograms of TMS derivatives of
18:1-ACP substrate (A) and product distributions for the castor desaturase triple mutant
T117R G188L D280K (B), and each of the single mutants T117R (C), G188L (D), and
D280K (E) reveals a novel fatty acid species labeled as peak 5. Product profile of wild-type
castor desaturase is included (F) as a control. Peak identities: Z18:1Δ9, (1); Z18:1Δ11, (2);
Z18:1Δ10 9OH, (3); E18:1Δ10 9OH, (4). www.plantphysiol.orgon October 31, 2020 - Published by Downloaded from Copyright © 2019 American Society of Plant Biologists. All rights reserved.
OSi(CH3)3
CH3-(CH2)6-CH-CH-CH-(CH2)7-CO2CH3
259
215
Fig. 2. The novel fatty acid product is 9,
10-dihydroxystearate. Comparison of
mass spectra of TMS derivatives of the
novel enzymatic product produced by the
castor desaturase T117R mutant (A) and an
authentic erythro 9,10 dihydroxy stearate
standard (C), and the fragmentation pattern
giving rise to the major ions at 215 and 259
AMU (B).
Rel
ativ
e A
bu
nd
ance
474
M
73
+
mass/charge
259215
(CH3)3SiO
Rel
ativ
e A
bu
nd
ance
474
M
73
+
259
mass/charge
215
A
B
C
www.plantphysiol.orgon October 31, 2020 - Published by Downloaded from Copyright © 2019 American Society of Plant Biologists. All rights reserved.
Fig. 3. The structural relationships of compounds discussed in this work. 1
Stearoyl ACP, showing two hydrogens at C-9, 10 that are removed by desaturase; 2
Oleoyl ACP, the product of stearoyl 9,10 desaturation; 3 Erythro-9(R) ,10 (R)-
dihydroxystearoyl ACP, the predicted product of 1 step direct oleate dihydroxylation;
4 Threo-9(S) ,10 (R)-dihydroxystearoyl ACP, a possible product of enzymatic 2 step
oleate epoxidation/hydrolysis sequence. www.plantphysiol.orgon October 31, 2020 - Published by Downloaded from Copyright © 2019 American Society of Plant Biologists. All rights reserved.
Fig. 4. The 9,10-dihydroxystearate produced by the
castor T117R mutant is solely in the erythro
configuration. Gas chromatograms of 9,10-dihydroxy-
stearates are compared for the reaction product of T117R
(A) to those of standards: threo configuration (B), the
erythro configuration (C), a mixture of the T117R
product and the threo standard (D), and the T117R
product and the erythro standard (E).
Det
ecto
r R
esp
on
se
Retention Time
A
B
C
E
D
www.plantphysiol.orgon October 31, 2020 - Published by Downloaded from Copyright © 2019 American Society of Plant Biologists. All rights reserved.
Fig. 5. Two potential schemes for the conversion of oleate to erythro 9,10
dihydroxystearate by a diiron-containing desaturase-dioxygenase. The initial bridged
hydroperoxo species in both mechanisms is inspired by large-scale multireference ab initio
calculations on a related enzyme (Chalupsky et al, 2014). www.plantphysiol.orgon October 31, 2020 - Published by Downloaded from Copyright © 2019 American Society of Plant Biologists. All rights reserved.
CH3-(CH2)6-CD2-CH-CH-(CH2)7-CO2CH3
CH3-(CH2)6-CD2-CH-CH-(CH2)7-CO2CH3
Fig. 6. Both hydroxyl oxygens of 9,10-dihydroxystearate are derived from molecular oxygen.
TMS derivatives of 9, 10-dihydroxystearate product from the castor desaturase T117R mutant
using 18:1 11-d2- substrate under air (A) or 18O2 shown in the diagram as O* (B).
Det
ecto
r R
esp
on
se
Retention Time
Det
ecto
r R
esp
on
se
Retention Time
Rel
ativ
e A
bu
nd
ance
480
M+
mass/charge
A
Rel
ativ
e A
bu
nd
ance
476
M+
mass/charge
217 259
261219
73
73
OSi(CH3)3
259
217
(CH3)3SiO
*OSi(CH3)3
261
219
(CH3)3SiO*
B
www.plantphysiol.orgon October 31, 2020 - Published by Downloaded from Copyright © 2019 American Society of Plant Biologists. All rights reserved.
355
357
355 359
359
355 357
A
B
C
356
356 360
360
Det
ecto
r R
esp
on
se
Retention Time Mass/Charge
Retention Time Mass/Charge
D
Det
ecto
r R
esp
on
se
355
357
356
Fig. 7. 9,10-dihydroxy stearate formation is the result of a
single dioxygenase reaction. Chromatograms and
corresponding mass spectra of acetonide derivatives of 9,10
dihydroxy stearate from reactions carried out under 16O2
(A), equimolar 16O2 and 18O2 (B), and 18O2 (C). Also
depicted is an authentic erythro 9,10 dihydroxy stearate
standard (D) along with a diagram of its fragmentation (E).
E
www.plantphysiol.orgon October 31, 2020 - Published by Downloaded from Copyright © 2019 American Society of Plant Biologists. All rights reserved.
1
Fig. 8. Upon prolonged incubation, the castor wild-type desaturase can convert 18:1 substrate to
erythro-9, 10-dihydroxystearate. Peak identities: Z18:1Δ9, (1); Z18:1Δ11, (2); Z18:1Δ10 9OH, (3);
E18:1Δ10 9OH; (4), and 9,10-dihydroxystearate (5).
Det
ecto
r R
esp
on
se
2
4
5
Retention Time (min.)
9.0 9.4 9.8 10.2
www.plantphysiol.orgon October 31, 2020 - Published by Downloaded from Copyright © 2019 American Society of Plant Biologists. All rights reserved.
Fig. 9. Low-level erythro-9, 10-dihydroxystearate is present in developing castor embryos. Gas
chromatogram of TMS derivatives of castor embryos (A) and the mass spectrum corresponding to peak
8 , i.e., 9,10-dihydroxystearate (B). Peak identities: 16:0 (1), 18:0 (2), 18:1D9 (3), 18:1D11 (4),
18:2D9,12 (5), 12-OH 18:1D9 (6), 18:3D9,12,15 (7), and 9, 10 OH 18:0 (8). C-E, GC peaks for TMS
derivatives of 9,10 dihydroxystearate from castor embryo (C), 9,10-dihydroxystearate from castor
developing embryos mixed with authentic threo-9,10-dihydroxystearate standard (D), and authentic
erythro-9,10-dihydroxystearate standard (E).
Rel
ativ
e A
bu
nd
ance
474
M
73
+
259
mass/charge
215
A
Det
ecto
r R
esp
on
se
1 2
3
4
5
6
7
B
Retention Time
8
Det
ecto
r R
esp
on
se
Retention Time
C
D
E
www.plantphysiol.orgon October 31, 2020 - Published by Downloaded from Copyright © 2019 American Society of Plant Biologists. All rights reserved.
Parsed CitationsArnold EV, Bohle DS (1996) Isolation and oxygenation reactions of nitrosylmyoglobins. Nitric Oxide, Pt B 269: 41-55
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Bai Y, McCoy JG, Levin EJ, Sobrado P, Rajashankar KR, Fox BG, Zhou M (2015) X-ray structure of a mammalian stearoyl-CoAdesaturase. Nature 524: 252-256
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Behrouzian B, Savile CK, Dawson B, Buist PH, Shanklin J (2002) Exploring the hydroxylation-dehydrogenation connection: novelcatalytic activity of castor stearoyl-ACP Delta(9) desaturase. J Am Chem Soc 124: 3277-3283.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Beremand PD, Hannapel DJ, Guerra DJ, Kuhn DN, Ohlrogge JB (1987) Synthesis, cloning, and expression in Escherichia coli of aspinach acyl carrier protein-I gene. Arch. Biochem. Biophys. 256: 90-100
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Bhar P, Reed DW, Covello PS, Buist PH (2012) Topological Study of Mechanistic Diversity in Conjugated Fatty Acid Biosynthesis.Angewandte Chemie-International Edition 51: 6686-6690
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Bloch K (1969) Enzymatic synthesis of monounsaturated fatty acids. Acc. Chem. Res. 2: 193-202Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Borrell M, Costas M (2017) Mechanistically Driven Development of an Iron Catalyst for Selective Syn-Dihydroxylation of Alkenes withAqueous Hydrogen Peroxide. Journal of the American Chemical Society 139: 12821-12829
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Broadwater JA, Whittle E, Shanklin J (2002) Desaturation and hydroxylation. Residues 148 and 324 of Arabidopsis FAD2, in addition tosubstrate chain length, exert a major influence in partitioning of catalytic specificity. J Biol Chem 277: 15613-15620
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Broun P, Shanklin J, Whittle E, Somerville C (1998) Catalytic plasticity of fatty acid modification enzymes underlying chemical diversityof plant lipids. Science 282: 1315-1317
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Buist PH (2004) Fatty acid desaturases: selecting the dehydrogenation channel. Nat Prod Rep 21: 249-262.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Cahoon EB, Lindqvist Y, Schneider G, Shanklin J (1997) Redesign of soluble fatty acid desaturases from plants for altered substratespecificity and double bond position. Proc. Natl. Acad. Sci. USA 94: 4872-4877
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Cahoon EB, Shanklin J (2000) Substrate-dependent mutant complementation to select fatty acid desaturase variants for metabolicengineering of plant seed oils. Proc Natl Acad Sci U S A 97: 12350-12355
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Cai YH, Yu XH, Liu Q, Liu CJ, Shanklin J (2018) Two clusters of residues contribute to the activity and substrate specificity of Fm1, abifunctional oleate and linoleate desaturase of fungal origin. Journal of Biological Chemistry 293: 19844-19853
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Chalupsky J, Rokob TA, Kurashige Y, Yanai T, Soomon EI, Rulisek L, Srnec M (2014) Reactivity of the Binuclear Non-Heme Iron ActiveSite of Delta(9) Desaturase Studied by Large-Scale Multireference Ab Initio Calculations. Journal of the American Chemical Society136: 15977-15991
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Crispino GA, Sharpless KB (1993) Enantioselective Synthesis of Juvenile Hormone-Iii in 3 Steps from Methyl Farnesoate. Synthesis-Stuttgart: 777-779
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title www.plantphysiol.orgon October 31, 2020 - Published by Downloaded from
Copyright © 2019 American Society of Plant Biologists. All rights reserved.
Edmondson DE, Juynh BH (1996) Diiron cluster intermediates in biological oxygen activation reactions. Inorg. Chimica Acta 252: 399-404
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Ensley BD, Gibson DT, Laborde AL (1982) Oxidation of Naphthalene by a Multicomponent Enzyme-System from Pseudomonas SpStrain Ncib9816. Journal of Bacteriology 149: 948-954
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Fox BG, Lyle KS, Rogge CE (2004) Reactions of the diiron enzyme stearoyl-acyl carrier protein desaturase. Acc Chem Res 37: 421-429Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Gherman BF, Baik MH, Lippard SJ, Friesner RA (2004) Dioxygen activation in methane monooxygenase: A theoretical study. Journal ofthe American Chemical Society 126: 2978-2990
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Guy JE, Whittle E, Kumaran D, Lindqvist Y, Shanklin J (2007) The crystal structure of the ivy Delta4-16:0-ACP desaturase revealsstructural details of the oxidized active site and potential determinants of regioselectivity. J Biol Chem 282:
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
: 19863-19871
Guy JE, Whittle E, Moche M, Lengqvist J, Lindqvist Y, Shanklin J (2011) Remote control of regioselectivity in acyl-acyl carrier protein-desaturases. Proc Natl Acad Sci U S A 108: 16594-16599
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Hudlicky T, Thorpe AJ (1996) Current status and future perspectives of cyclohexadiene-cis-diols in organic synthesis: Versatileintermediates in the concise design of natural products. Chemical Communications: 1993-2000
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Karlsson A, Parales JV, Parales RE, Gibson DT, Eklund H, Ramaswamy S (2003) Crystal structure of naphthalene dioxygenase: Side-onbinding of dioxygen to iron. Science 299: 1039-1042
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
King G (1942) The dihydroxystearitc acid of castor oil - Its constitution and structural relationship to the 9 10-dihydroxystearic acids mp's 132 degrees and 95 degrees. Journal of the Chemical Society: 387-391
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Komor AJ, Rivard BS, Fan RX, Guo YS, Que L, Lipscomb JD (2017) CmII N-Oxygenase Catalyzes the Final Three Steps inChloramphenicol Biosynthesis without Dissociation of Intermediates. Biochemistry 56: 4940-4950
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Lee M, Lenman M, Banas A, Bafor M, Singh S, Schweizer M, Nilsson R, Liljenberg C, Dahlqvist A, Gummeson PO, Sjodahl S, Green A,Stymne S (1998) Identification of non-heme diiron proteins that catalyze triple bond and epoxy group formation. Science 280: 915-918
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
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