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Letter
Stereospecificity of enoylreductase domains from modular polyketide synthases Luyun Zhang, Junjie Ji, Meijuan Yuan, Yuanyuan Feng, Lei Wang, Zixin Deng, Linquan Bai, and Jianting Zheng ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.7b00982 • Publication Date (Web): 13 Feb 2018 Downloaded from http://pubs.acs.org on February 14, 2018
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Stereospecificity
2
Polyketide Synthases
3
Luyun Zhang,a,c Junjie Ji,b,c Meijuan Yuan,a Yuanyuan Feng,a Lei Wang,a Zixin Deng,a Linquan
4
Bai,a Jianting Zheng*a
5
a
6
Shanghai Jiao Tong University, Shanghai 200240, China
7
b
8
Academy of Sciences, Beijing, 100190, PR China
9
c
10
of
Enoylreductase
Domains
from
Modular
State Key Laboratory of Microbial Metabolism, and School of Life Sciences and Biotechnology,
National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese
Luyun Zhang and Junjie Ji contributed equally to this work
*Correspondence:
[email protected] 11
1
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ABSTRACT
13
An enoylreductase (ER) domain of a polyketide synthase module recruiting a methylmalonate
14
extender unit sets the C2 methyl branch to either S or R configuration during processing of a
15
polyketide intermediate carried by an acyl carrier protein (ACP) domain. In the present study,
16
pantetheine- and ACP-bound trans-2-methylcrotonyl substrate surrogates were used to scrutinize
17
the stereospecificity of the ER domains. The pantetheine-bound thioester was reduced to mixtures
18
of both 2R and 2S products, whereas the expected 2S epimer was almost exclusively generated
19
when the cognate ACP-bound substrate surrogate was utilized. The analogous incubation of an ER
20
with the substrate surrogate carried by a noncognate ACP significantly increased the generation of
21
the unexpected 2R epimer, highlighting the dependence of stereospecificity on proper
22
protein-protein interactions between ER and ACP domains. The ER mutant assays revealed the
23
involvement of the conserved tyrosine and lysine in stereocontrol. Taken together, these results
24
expand the current understanding of the ER stereochemistry and help in the engineering of
25
modular PKSs.
26
2
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Polyketides are a large group of natural products with diverse bioactivities, including antibacterial,
28
antifungal, antitumor and immunosuppressive properties.1, 2 Many polyketides are assembled by
29
modular polyketide synthases (PKSs) from coenzyme A (CoA) activated carboxylic acids in a
30
manner resembling fatty acid biosynthesis. However, a considerably wider variety of carboxylic
31
acids can be utilized by modular PKSs to generate enormous chemical diversity. A ketosynthase
32
(KS), an acyltransferase (AT), and an acyl carrier protein (ACP) constitute a minimal module that
33
is responsible for the elongation of polyketide chain, whereas the combination of ketoreductase
34
(KR), dehydratase (DH), and enoylreductase (ER) domains in a module controls the final
35
oxidation state of extender unit newly added to the growing polyketide chain. The resulting
36
polyketides are stereochemically dense due to the methyl-bearing chiral centers arising from the
37
incorporation of methylmalonate extender units and the hydroxyl-bearing chiral centers introduced
38
by KRs.3, 4
39
The past two decades have seen considerable progress in understanding the way that modular PKSs
40
control the stereochemistry of methyl and hydroxyl substituents and in relating the stereochemical
41
outcomes to the intrinsic specificity of the catalytic domains. An AT recruits exclusively the
42
(2S)-isomer of methylmalonyl-CoA, while a KS catalyzes the decarboxylative condensation of
43
extender
44
(2R)-2-methyl-3-ketoacyl-ACP intermediate.5, 6 Significant efforts have been made to determine the
45
stereochemistry of KR-catalyzed reactions, since they determine the configuration of both C2 methyl
46
and C3 hydroxyl of polyketide chains.7 In vivo studies demonstrate that swapping KR domains between
47
different modular PKSs results in the generation of polyketides with altered stereochemistry,
48
suggesting that stereospecificity is an intrinsic property of KR domains and is transferable.8 Structural
49
analysis reveals the precise domain boundaries and facilitates the dissection of a complete module into
50
discrete active domains.9 In combination with the development of robust methods to assign the
51
stereochemistry of the reduced products, the catalytic activities of individual KRs have been
52
investigated intensively in vitro.10 Divergent sequence motifs are correlated to the stereospecificity of
53
KRs and used to predict the absolute configuration of polyketide products.11,
54
residues in these motifs can switch the stereochemical outcome of KRs at least in vitro.13
55
unit
with
the
inversion
of
configuration
at
C2
position
12
to
generate
Alteration of the
By contrast, the stereospecificity of ER domains of the modular PKSs is not well-studied. 3
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Analogous to their counterparts in fatty acid synthases (FASs), ERs of modular PKSs belong to
57
the medium chain NAD(P)H-dependent dehydrogenase/reductase (MDR) superfamily.14 In a
58
module that recruits methylmalonyl-CoA, the ER reduces the carbon-carbon double bond of
59
2-enoyl intermediate and simultaneously sets the methyl branch at C2 position in either R or S
60
configuration (Figure 1). In vivo functional assays within the context of a complete module reveal
61
a correlation between a unique residue in the ER active site and chirality of the generated methyl
62
branch.15 In an ER producing an S-configured methyl branch, this residue is systematically
63
conserved as tyrosine, while in the ER domains producing an R-configured methyl branch, valine
64
(occasionally alanine or phenylalanine) is observed at this position. A single mutation of tyrosine
65
to valine in the ER from the module 4 of erythromycin PKS (EryER4) switches the configuration
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of the corresponding methyl branch from S to R. We have solved a KR-ER didomain structure
67
from the second module of spinosyn PKS.16 This didomain structure defines the precise boundary
68
of ER domains and facilitates the expression of SpnER2 as a discrete active domain. The
69
inspection of the structure reveals that a lysine and an aspartic acid are situated near nicotinamide
70
ring of the NADPH cofactor besides the fingerprint tyrosine of S-type ERs. However, no single
71
mutation alone abolishes the in vitro activity of SpnER2 completely, consistent with the in vivo
72
mutational study on the ER domain from module 13 of rapamycin PKS (RapER13), which also
73
fails to reveal a single residue that is essential for activity.17
74
In this study, we report the in vitro characterization of the stereospecificity of ER domains from
75
the modular PKSs. The stereochemical control of isolated KRs has been extensively assayed, and
76
it revealed the potential of several KRs as catalysts for the stereospecific reduction of ketones in
77
organic synthesis; but, to date, SpnER2 is the only isolated ER domain of the modular PKS
78
characterized in vitro.16 To investigate the in vitro stereospecificity of discrete ER domains, two
79
additional ERs from the fourth modules of erythromycin and pikromycin PKSs were also
80
expressed in Escherichia coli BL21 (DE3). The boundaries of ER domains were chosen using
81
sequence alignment with SpnER2, of which two β-strands (β1 and β9) in the substrate-binding
82
subdomain were defined as the N-terminus and C-terminus respectively. A high level of overall
83
sequence identity was observed between SpnER2 and EryER4 (54%), SpnER2 and PikER4 (65%),
84
and EryER4 and PikER4 (56%). All these ER domains were expressed in a soluble form with 4
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average yields of more than 5 mg/L. SpnER2 has been previously shown to reduce
86
trans-α,β-unsaturated crotonyl-pantetheine. Although there is no methyl branch on α-carbon of the
87
natural substrate trans-(5S)-hydroxyhept-2-enoyl-SpnACP2 thioester,18 SpnER2 could indeed
88
reduce this substrate carrying an α-methyl branch (Figure 2). The high-performance liquid
89
chromatography (HPLC) analysis of the reaction mixtures showed the generation of a reduced
90
product, which was verified to be 2-methylbutyryl-pantetheine by the mass spectrum (HR-ESI-MS
91
m/z 385.1767 observed; calculated for C16H30N2NaO5S 385.1768 [M+Na]+). As shown in Figure
92
2B, EryER4 and PikER4 could also reduce the trans-2-methylcrotonyl-pantetheine substrate
93
surrogate (Figure 2B). N-acetyl cysteamine (NAC) and coenzyme A (CoA) derived thioesters
94
were also synthesized, whereas the reduced product was not observed when they were incubated
95
with these ERs in the same reaction condition (Figure S1). It has been reported that an ER domain
96
from a highly reducing iterative fungal PKS also prefers pantetheine-bound substrate to the
97
corresponding NAC thioesters.19
98
A convenient and sensitive protocol to resolve the two isomers of 2-methylbutyric acid by chiral
99
gas chromatographic/mass spectrometric (GC/MS) method has been developed previously and
100
used to determine the in vitro stereospecificity of the loading AT from avermectin PKS toward the
101
racemic 2-methylbutyryl-NAC thioester.20 The direct comparison of the experimentally measured
102
retention times obtained using a β-DEX 120 capillary column, as well as the corresponding mass
103
spectra with those of the authentic standards, could enable the determination of the absolute
104
configuration and the relative yield of each 2-methylbutyric acid isomer (Figure 3). To establish
105
the stereochemical course of ER-catalyzed reduction, the reactions were quenched after 48 hours
106
of incubation by the addition of an aqueous base. Following acidification and extraction, the
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resulting 2-methylbutyric acid was subjected to chiral GC/MS analysis to assign the
108
stereochemistry of C2 methyl branch. SpnER2 contains a tyrosine at the corresponding position of
109
the active site, suggesting its function as an S-type ER. However, the expected 2S isomer only
110
constituted ~60% of all reduced products (Figure 3B). Due to the absence of a C2 methyl branch
111
in the natural substrate, SpnER2 may not be exposed to the corresponding evolutionary pressure.
112
We sought to determine the stereochemical outcome of the reactions catalyzed by the recombinant
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EryER4 and PikER4, which incorporated S-configured methyl substituents into erythromycin and 5
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pikromycin respectively (Figure 2A), consistent with the conserved tyrosine at their active sites.
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Intriguingly, incubation of trans-2-methylcrotonyl-pantetheine with EryER4 and PikER4 also
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generated both 2S and 2R products (Figure 3B). More than 60% substrate still remained after the
117
reactions were incubated for 48 hours (Fig. S2). We determined the R/S ratios at 12, 24, 36 and 48
118
hours. Modest fluctuations were revealed, suggesting that the R- and S-isomers were likely
119
produced at similar rates.
120
The effects of ACPs on the stereospecificity of ERs are assayed, since all intermediates are
121
tethered
122
trans-2-methylcrotonyl-ACPs were enzymatically synthesized from the corresponding CoA
123
thioester by the promiscuous Sfp phosphopantetheinyl transferase from Bacillus subtilis.21 The
124
reduced product was treated with trypsin protease before base-catalyzed hydrolysis to increase the
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release of 2-methylbutyric acid from the phosphopantetheine arm of ACP domain. It has been
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reported previously that the ACP-bound thioesters are more stable compared with their
127
corresponding
128
phosphopantetheinate-tethered substrate by accommodating it in a cavity has been proposed.10
129
Notably, all three ER domains retained their intrinsic stereospecificity toward the cognate
130
ACP-bound substrate surrogates and predominantly yielded the expected (2S)-methylbutyryl-ACP
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(Figure 3C). Analogous incubations were performed using ERs and non-cognate ACPs (Table S1).
132
EryER4 and PikER4 reduced the SpnACP2-bound substrate to the expected 2S product, whereas
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significant amount of unexpected (2R)-methylbutyryl-ACPs were produced by the paired SpnER2
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+ EryACP4, SpnER2 + PikACP4, EryER4 + PikACP4, and PikER4 + EryACP4, highlighting the
135
dependence of ER stereospecificity on proper protein-protein interactions between ER and ACP
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domains. Conversely, stereochemistry of the reduced 2-methyl-3-hydroxyacyl-ACP was directly
137
correlated with the specificity of relevant KR domain, independent of which the ACP was used to
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generate the initial 2-methyl-3-ketoacyl-ACP intermediate.22 Crystal structures of an AT–ACP
139
complex from the vicenistatin PKS and a curacin β-branching enzyme in complex with ACP
140
indicate that the ACP surface of αII, loop II and αIII interacts with partner enzymes.23, 24 The
141
SpnACP2 surface for ER interaction is likely more promiscuous compared with EryACP4 and
142
PikACP4. Pairwise sequence alignments of three ACPs suggested a high degree of sequence
to
ACPs
NAC
during
the
thioesters.
processing
A
hypothesis
of
polyketide
that
an
intermediates.
ACP
stabilizes
The
the
6
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identity (SpnACP2-EryACP4 51%, SpnER2-PikACP4 54%, and EryACP4-PikACP4 58%) but
144
revealed two conserved aspartate residues on the tentative contact surface of EryACP4 and
145
PikACP4. The distinctive surface shape and charge distribution of ACPs may contribute to their
146
specificity against ERs (Figure S3). A structure of ER-ACP complex will improve our
147
understanding of the basis of ACP recognition for ER stereochemistry.
148
The mechanism of ER-catalyzed reduction assumes a direct transfer of a hydride from NADPH
149
to the C3 position of the enoyl substrate followed by stereospecific protonation at the C2 position,
150
which determines the configuration of methyl substituent.15 A recently proposed ene mechanism
151
involves the formation of a transient covalent NADPH-substrate adduct (‘C2-ene adduct’) in the
152
transfer of the hydride equivalent.25 The C2-ene adduct is further converted to the consensus
153
enolate intermediate, which accepts a proton to form the reduced product. In either case, a proper
154
proton donor at the active site is indispensable and determines the configuration of C2 methyl
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branch in the reductions catalyzed by ERs of modular PKSs. The structure of SpnER2 reveals that
156
three conserved residues Y241, K422 and D444 are ~6 Å from the nicotinamide hydride (Figure
157
4). We sought to identify the effects of these residues on the stereospecificity of ERs. K422 and
158
D444 were mutated to alanine to determine the contributions of the side chains, while Y241 was
159
mutated to phenylalanine to abolish its potential function as a proton donor. The D444A of
160
SpnER2 produced the 2S product exclusively, whereas Y241F and K422A mutants lost the
161
stereospecificity toward the ACP-bound substrate thioester and produced a mixture of both 2S and
162
2R products (Table S2). Similar results were obtained for the corresponding mutants of EryER4
163
and PikER4, suggesting that the conserved tyrosine and lysine are involved in the stereocontrol of
164
ER domains. In vivo functional assay of EryER4 shows that the single mutation of tyrosine to
165
valine switches the configuration of the resulting methyl branch from S to R completely.15
166
Surprisingly, both S-type (~50%) and R-type (~50%) products were generated in vitro by the
167
tyrosine to valine mutants of SpnER2, EryER4, and PikER4 (Table S2). In addition to the
168
relatively shorter acyl chain compared with the native substrate, other catalytic domains in the
169
PKS modules may also affect the stereospecificity of ERs. More sensitive experiments are
170
necessary to clarify the exact role of each catalytic residue.
171
In summary, understanding the molecular basis for the stereospecificity of modular PKSs is 7
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crucial for production of novel polyketides by protein engineering. In this study, pantetheine and
173
ACP bound trans-2-methylcrotonyl substrate surrogates were incubated with discrete ERs from
174
different modular PKSs in the presence of NADPH. Following the release of 2-methylbutyric acid
175
by base-catalyzed hydrolysis, the stereochemistry of the reduced product was assigned by chiral
176
GC/MS. The dependence of ER stereospecificity on cognate ACP was revealed with this method.
177
The effects of catalytic residues on the stereospecificity of ERs were investigated in combination
178
with site-specific mutagenesis. Taken together, the results presented in this report expand the
179
current understanding of ER stereochemistry and could facilitate the rational engineering of
180
modular PKSs.
181 182
METHODS
183
See the Supporting Information for a detailed description of the experimental methods.
184
ASSOCIATED CONTENT
185
Supporting Information
186
The supporting information is available free of charge via the internet at http://pubs.acs.org.
187
Includes supporting tables, supporting figures, and detailed methods that describe expression and
188
purification of ERs; site-directed mutagenesis; synthesis of trans-2-methylcrotonyl-CoA;
189
reduction of thioester substrate and trans-2-methylcrotonyl-ACP; chiral GC/MS; and homology
190
modeling.
191 192
AUTHOR INFORMATION
193
Corresponding Author
194
*E-mail:
[email protected] 195
ORCID
196
Jianting Zheng: 0000-0003-1250-3556 8
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Notes
199
The authors declare no competing financial interest.
200 201
ACKNOWLEDGMENTS
202
This work was supported in part by the National Natural Science Foundation of China (31370101,
203
31570056, 31770068), the National Program on Key Basic Research Project (973 program,
204
2013CB734002), the State Key Laboratory of Pathogen and Biosecurity (Academy of Military
205
Medical Science, SKLPBS1526), the Fundamental Research Funds for the Central Universities,
206
and the Global Talents Recruitment Program. The authors acknowledge K. Yang of Institute of
207
Microbiology, Chinese Academy of Sciences for providing Saccharopolyspora spinosa and the
208
core facility of state key laboratory of microbial metabolism for assistance in GC/MS.
209 210
References
211
1. Wong, F. T., and Khosla, C. (2012) Combinatorial biosynthesis of polyketides--a perspective, Curr
212
Opin Chem Biol 16, 117-123.
213
2. Robbins, T., Liu, Y. C., Cane, D. E., and Khosla, C. (2016) Structure and mechanism of assembly line
214
polyketide synthases, Curr Opin Struct Biol 41, 10-18.
215
3. Kwan, D. H., and Schulz, F. (2011) The stereochemistry of complex polyketide biosynthesis by
216
modular polyketide synthases, Molecules 16, 6092-6115.
217
4. Weissman, K. J. (2017) Polyketide stereocontrol: a study in chemical biology, Beilstein J Org Chem 13,
218
348-371.
219
5. Marsden, A. F., Caffrey, P., Aparicio, J. F., Loughran, M. S., Staunton, J., and Leadlay, P. F. (1994)
220
Stereospecific acyl transfers on the erythromycin-producing polyketide synthase, Science 263,
221
378-380. 9
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ACS Chemical Biology
222
6. Weissman, K. J., Timoney, M., Bycroft, M., Grice, P., Hanefeld, U., Staunton, J., and Leadlay, P. F.
223
(1997) The molecular basis of Celmer's rules: the stereochemistry of the condensation step in chain
224
extension on the erythromycin polyketide synthase, Biochemistry 36, 13849-13855.
225
7. Zheng, J., and Keatinge-Clay, A. (2013) The status of type I polyketide synthase ketoreductases, Med.
226
Chem. Commun. 4, 34-40.
227
8. Kao, C. M., McPherson, M., McDaniel, R. N., Fu, H., Cane, D. E., and Khosla, C. (1998) Alcohol
228
Stereochemistry in Polyketide Backbones Is Controlled by the β-Ketoreductase Domains of Modular
229
Polyketide Synthases, J Am Chem Soc 120, 2478-2479.
230
9. Keatinge-Clay, A. T., and Stroud, R. M. (2006) The structure of a ketoreductase determines the
231
organization of the beta-carbon processing enzymes of modular polyketide synthases, Structure 14,
232
737-748.
233
10. Castonguay, R., He, W., Chen, A. Y., Khosla, C., and Cane, D. E. (2007) Stereospecificity of
234
ketoreductase domains of the 6-deoxyerythronolide B synthase, J Am Chem Soc 129, 13758-13769.
235
11. Caffrey, P. (2003) Conserved amino acid residues correlating with ketoreductase stereospecificity
236
in modular polyketide synthases, Chembiochem 4, 654-657.
237
12. Reid, R., Piagentini, M., Rodriguez, E., Ashley, G., Viswanathan, N., Carney, J., Santi, D. V.,
238
Hutchinson, C. R., and McDaniel, R. (2003) A model of structure and catalysis for ketoreductase
239
domains in modular polyketide synthases, Biochemistry 42, 72-79.
240
13. Baerga-Ortiz, A., Popovic, B., Siskos, A. P., O'Hare, H. M., Spiteller, D., Williams, M. G., Campillo, N.,
241
Spencer, J. B., and Leadlay, P. F. (2006) Directed mutagenesis alters the stereochemistry of catalysis by
242
isolated ketoreductase domains from the erythromycin polyketide synthase, Chem Biol 13, 277-285.
243
14.
244
dehydrogenase/reductase gene and protein families : the MDR superfamily, Cell Mol Life Sci 65,
245
3879-3894.
246
15. Kwan, D. H., Sun, Y., Schulz, F., Hong, H., Popovic, B., Sim-Stark, J. C., Haydock, S. F., and Leadlay, P. F.
247
(2008) Prediction and manipulation of the stereochemistry of enoylreduction in modular polyketide
248
synthases, Chem Biol 15, 1231-1240.
249
16. Zheng, J., Gay, D. C., Demeler, B., White, M. A., and Keatinge-Clay, A. T. (2012) Divergence of
250
multimodular polyketide synthases revealed by a didomain structure, Nat Chem Biol 8, 615-621.
Persson,
B.,
Hedlund,
J.,
and
Jornvall,
H.
(2008)
Medium-
and
short-chain
10
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Page 12 of 16
251
17. Kwan, D. H., and Leadlay, P. F. (2010) Mutagenesis of a modular polyketide synthase
252
enoylreductase domain reveals insights into catalysis and stereospecificity, ACS Chem Biol 5, 829-838.
253
18. Waldron, C., Matsushima, P., Rosteck, P. R., Jr., Broughton, M. C., Turner, J., Madduri, K., Crawford,
254
K. P., Merlo, D. J., and Baltz, R. H. (2001) Cloning and analysis of the spinosad biosynthetic gene cluster
255
of Saccharopolyspora spinosa, Chem Biol 8, 487-499.
256
19. Roberts, D. M., Bartel, C., Scott, A., Ivison, D., Simpson, T. J., and Cox, R. J. (2017) Substrate
257
selectivity of an isolated enoyl reductase catalytic domain from an iterative highly reducing fungal
258
polyketide synthase reveals key components of programming, Chem Sci 8, 1116-1126.
259
20. Wang, F., Wang, Y., Ji, J., Zhou, Z., Yu, J., Zhu, H., Su, Z., Zhang, L., and Zheng, J. (2015) Structural
260
and functional analysis of the loading acyltransferase from avermectin modular polyketide synthase,
261
ACS Chem Biol 10, 1017-1025.
262
21. Quadri, L. E. N., Weinreb, P. H., Lei, M., Nakano, M. M., Zuber, P., and Walsh, C. T. (1998)
263
Characterization of Sfp, a Bacillus subtilis phosphopantetheinyl transferase for peptidyl carrier protein
264
domains in peptide synthetases, Biochemistry 37, 1585-1595.
265
22. Ostrowski MP, Cane DE, Khosla C. (2016) Recognition of acyl carrier proteins by ketoreductases in
266
assembly line polyketide syntheses, J Antibiot 69,507-510.
267
23. Miyanaga, A., Iwasawa, S., Shinohara, Y., Kudo, F., and Eguchi, T. (2016) Structure-based analysis of
268
the molecular interactions between acyltransferase and acyl carrier protein in vicenistatin biosynthesis,
269
Proc Natl Acad Sci U S A 113, 1802-1807.
270
24. Maloney, F. P., Gerwick, L., Gerwick, W. H., Sherman, D. H., and Smith, J. L. (2016) Anatomy of the
271
beta-branching enzyme of polyketide biosynthesis and its interaction with an acyl-ACP substrate, Proc
272
Natl Acad Sci U S A 113, 10316-10321.
273
25. Rosenthal, R. G., Vogeli, B., Quade, N., Capitani, G., Kiefer, P., Vorholt, J. A., Ebert, M. O., and Erb, T.
274
J. (2015) The use of ene adducts to study and engineer enoyl-thioester reductases, Nat Chem Biol 11,
275
398-400.
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Figure 1. An ER domain of a modular polyketide synthase sets the α-methyl substituent to either S or R configuration in the reduction of double bond. 66x26mm (300 x 300 DPI)
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Figure 2. The ER-catalyzed reduction of trans-2-methylcrotonyl-pantetheine. (A) Structure of spinosyn, erythromycin and pikromycin. The methyl substituents of which the stereochemistry is controlled by EryER4 and PikER4 respectively are labeled by red asterisk. SpnER2 catalyzes the reduction of polyketide intermediate without α-methyl branch in the biosynthesis of spinosyn. (B) All three tested ER can reduce the pantetheine-bound substrate surrogate. 140x61mm (300 x 300 DPI)
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Figure 3. Stereospecificity of ER domains. (A) The two isomers of 2-methylbutyric acid can be resolved by chiral gas chromatography using a β-DEX 120 capillary column and show identical fragmentation patterns in the mass spectra. (B) Both 2S and 2R products were produced in the reduction reactions of trans-2methylcrotonyl-pantetheine catalyzed by SpnER2, EryER4 and PikER4. (C) Each ER almost exclusively reduced its cognate trans-2-methylcrotonyl-ACP to the expected 2S product. 66x87mm (300 x 300 DPI)
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Figure 4. The active site of SpnER2. (A) The structure shows SpnER2 active site. Conserved catalytic residues (Y241, K422 and D444) and NADP+ coenzyme are shown as sticks. (B) Sequence alignment of SpnER2, EryER4 and PikER4. Residues are number according to SpnER2. The catalytic residues are labeled by red asterisk. 109x80mm (300 x 300 DPI)
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