Stereospecificity of Enoylreductase Domains from Modular Polyketide

Feb 13, 2018 - An enoylreductase (ER) domain of a polyketide synthase module recruiting a methylmalonate extender unit sets the C2 methyl branch to ei...
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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

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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

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(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

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(KR), dehydratase (DH), and enoylreductase (ER) domains in a module controls the final

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oxidation state of extender unit newly added to the growing polyketide chain. The resulting

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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

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The past two decades have seen considerable progress in understanding the way that modular PKSs

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control the stereochemistry of methyl and hydroxyl substituents and in relating the stereochemical

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outcomes to the intrinsic specificity of the catalytic domains. An AT recruits exclusively the

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(2S)-isomer of methylmalonyl-CoA, while a KS catalyzes the decarboxylative condensation of

43

extender

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(2R)-2-methyl-3-ketoacyl-ACP intermediate.5, 6 Significant efforts have been made to determine the

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stereochemistry of KR-catalyzed reactions, since they determine the configuration of both C2 methyl

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and C3 hydroxyl of polyketide chains.7 In vivo studies demonstrate that swapping KR domains between

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different modular PKSs results in the generation of polyketides with altered stereochemistry,

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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

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discrete active domains.9 In combination with the development of robust methods to assign the

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stereochemistry of the reduced products, the catalytic activities of individual KRs have been

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investigated intensively in vitro.10 Divergent sequence motifs are correlated to the stereospecificity of

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KRs and used to predict the absolute configuration of polyketide products.11,

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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

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the medium chain NAD(P)H-dependent dehydrogenase/reductase (MDR) superfamily.14 In a

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module that recruits methylmalonyl-CoA, the ER reduces the carbon-carbon double bond of

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2-enoyl intermediate and simultaneously sets the methyl branch at C2 position in either R or S

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configuration (Figure 1). In vivo functional assays within the context of a complete module reveal

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a correlation between a unique residue in the ER active site and chirality of the generated methyl

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branch.15 In an ER producing an S-configured methyl branch, this residue is systematically

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conserved as tyrosine, while in the ER domains producing an R-configured methyl branch, valine

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(occasionally alanine or phenylalanine) is observed at this position. A single mutation of tyrosine

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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

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from the second module of spinosyn PKS.16 This didomain structure defines the precise boundary

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of ER domains and facilitates the expression of SpnER2 as a discrete active domain. The

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inspection of the structure reveals that a lysine and an aspartic acid are situated near nicotinamide

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ring of the NADPH cofactor besides the fingerprint tyrosine of S-type ERs. However, no single

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mutation alone abolishes the in vitro activity of SpnER2 completely, consistent with the in vivo

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mutational study on the ER domain from module 13 of rapamycin PKS (RapER13), which also

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fails to reveal a single residue that is essential for activity.17

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In this study, we report the in vitro characterization of the stereospecificity of ER domains from

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the modular PKSs. The stereochemical control of isolated KRs has been extensively assayed, and

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it revealed the potential of several KRs as catalysts for the stereospecific reduction of ketones in

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organic synthesis; but, to date, SpnER2 is the only isolated ER domain of the modular PKS

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characterized in vitro.16 To investigate the in vitro stereospecificity of discrete ER domains, two

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additional ERs from the fourth modules of erythromycin and pikromycin PKSs were also

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expressed in Escherichia coli BL21 (DE3). The boundaries of ER domains were chosen using

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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

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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

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trans-α,β-unsaturated crotonyl-pantetheine. Although there is no methyl branch on α-carbon of the

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natural substrate trans-(5S)-hydroxyhept-2-enoyl-SpnACP2 thioester,18 SpnER2 could indeed

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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

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2B, EryER4 and PikER4 could also reduce the trans-2-methylcrotonyl-pantetheine substrate

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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

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from a highly reducing iterative fungal PKS also prefers pantetheine-bound substrate to the

97

corresponding NAC thioesters.19

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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

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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

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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

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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

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constituted ~60% of all reduced products (Figure 3B). Due to the absence of a C2 methyl branch

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in the natural substrate, SpnER2 may not be exposed to the corresponding evolutionary pressure.

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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

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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

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produced at similar rates.

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The effects of ACPs on the stereospecificity of ERs are assayed, since all intermediates are

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tethered

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trans-2-methylcrotonyl-ACPs were enzymatically synthesized from the corresponding CoA

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thioester by the promiscuous Sfp phosphopantetheinyl transferase from Bacillus subtilis.21 The

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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

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corresponding

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phosphopantetheinate-tethered substrate by accommodating it in a cavity has been proposed.10

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Notably, all three ER domains retained their intrinsic stereospecificity toward the cognate

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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).

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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

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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

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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

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complex from the vicenistatin PKS and a curacin β-branching enzyme in complex with ACP

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indicate that the ACP surface of αII, loop II and αIII interacts with partner enzymes.23, 24 The

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SpnACP2 surface for ER interaction is likely more promiscuous compared with EryACP4 and

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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

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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

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understanding of the basis of ACP recognition for ER stereochemistry.

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The mechanism of ER-catalyzed reduction assumes a direct transfer of a hydride from NADPH

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to the C3 position of the enoyl substrate followed by stereospecific protonation at the C2 position,

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which determines the configuration of methyl substituent.15 A recently proposed ene mechanism

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involves the formation of a transient covalent NADPH-substrate adduct (‘C2-ene adduct’) in the

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transfer of the hydride equivalent.25 The C2-ene adduct is further converted to the consensus

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enolate intermediate, which accepts a proton to form the reduced product. In either case, a proper

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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

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SpnER2 produced the 2S product exclusively, whereas Y241F and K422A mutants lost the

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stereospecificity toward the ACP-bound substrate thioester and produced a mixture of both 2S and

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2R products (Table S2). Similar results were obtained for the corresponding mutants of EryER4

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and PikER4, suggesting that the conserved tyrosine and lysine are involved in the stereocontrol of

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ER domains. In vivo functional assay of EryER4 shows that the single mutation of tyrosine to

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valine switches the configuration of the resulting methyl branch from S to R completely.15

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Surprisingly, both S-type (~50%) and R-type (~50%) products were generated in vitro by the

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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

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PKS modules may also affect the stereospecificity of ERs. More sensitive experiments are

170

necessary to clarify the exact role of each catalytic residue.

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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

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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

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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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