Discovery of a Switch Between Prelog and Anti-Prelog Reduction

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Discovery of a Switch Between Prelog and antiPrelog Reduction Toward Halogen-Substituted Acetophenones in Short-Chain Dehydrogenase/Reductases Fengyu Qin, Bin Qin, Wenhe Zhang, Yalin Liu, Xin Su, Tianhui Zhu, Jingping Ouyang, Jiyang Guo, Yuxin Li, Feiting Zhang, Jun Tang, Xian Jia, and Song You ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.8b00807 • Publication Date (Web): 23 May 2018 Downloaded from http://pubs.acs.org on May 23, 2018

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ACS Catalysis

Discovery of a Switch Between Prelog and anti-Prelog Reduction Toward Halogen-Substituted Acetophenones in Short-Chain Dehydrogenase/Reductases

Fengyu Qin,†,‡ Bin Qin,*,¶,‡ Wenhe Zhang,† Yalin Liu,† Xin Su,† Tianhui Zhu,† Jingping Ouyang,§ Jiyang Guo,† Yuxin Li,† Feiting Zhang,† Jun Tang,† Xian Jia,*,§ and Song You*,†

†

School of Life Sciences and Biopharmaceutical Sciences, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenhe, Shenyang 110016, China

¶

Wuya College of Innovation, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenhe, Shenyang 110016, China §

School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenhe, Shenyang 110016, China

ACS Paragon Plus Environment

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ABSTRACT The application of ketoreductases based biocatalytic reduction to access optically pure Prelog or anti-Prelog alcohols offers a valuable approach for asymmetric synthesis. Despite this, control the stereopreferences of ketoreductases as desired remains challenging since natural ketoreductases usually display Prelog preference and it is difficult to transfer the knowledge from engineered anti-Prelog ketoreductases to the others. Here, we present the discovery of a switch between Prelog and anti-Prelog reduction toward halogen-substituted acetophenones in six short-chain dehydrogenase/reductases (SDRs). Through carefully analyzing the structural information and multiple-sequence alignment of several reported SDRs with Prelog or antiPrelog stereopreference, the key residues that might control their stereopreferences were identified using Lactobacillus fermentum short-chain dehydrogenase/reductase 1 (LfSDR1) as the starting enzyme. Protein engineering at these positions of LfSDR1 could improve its antiPrelog stereoselectivity or switch its stereopreference to Prelog. Moreover, the knowledge obtained from LfSDR1 could be further transferred to other five SDRs (four mined SDRs and one reported SDR) that have 21-48% sequence identities with LfSDR1. The stereopreferences of these SDRs were able to be switched either from anti-Prelog to Prelog or from Prelog to antiPrelog by mutagenesis at related positions. In addition, further optimization of LfSDR1 can access stereo-complementary reduction of several halogen-substituted acetophenones with high stereoselectivity (up to >99%), resulting in some valuable chiral alcohols for synthesis of pharmaceutical agents.

KEYWORDS: short-chain dehydrogenase/reductases, protein engineering, halogen-substituted acetophenones, asymmetric reduction, stereopreference switch, biocatalysis


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ACS Catalysis

INTRODUCTION Ketoreductases are known to be powerful biocatalysts in asymmetric synthesis since their reduction of prochiral ketones to corresponding chiral alcohols usually display high stereoselectivity.1-9 Promoted by the utilization of protein engineering to further improve activity and selectivity, ketoreductases are being widely used in pharmaceutical synthesis and organic synthesis.1-9 For instance, several industrial-scale processes that use ketoreductases-catalysis as key steps for asymmetric synthesis of blockbuster drugs, such as atorvastatin (Lipitor), montelukast (Singulair), crizotinib (Xalkori) and so on, have been developed.4,8 As well known, the asymmetric reduction of prochiral carbonyl compounds by most ketoreductases generally follows Prelog’s rule, which would produce Prelog products10. However, both Prelog alcohols and anti-Prelog alcohols could be used as building blocks in pharmaceutical and fine chemical synthesis, thus, ketoreductases following Prelog’s or antiPrelog’s rules are equally desired. Since ketoreductases with anti-Prelog stereoselectivity (such as LbADH,11 LkADH,12 and so on) are relatively rare in nature,10 several efforts have been tried to engineer Prelog ketoreductases into anti-Prelog ketoreductases. For instance, Phillips and coworkers reversed the stereopreference of TeSADH from Prelog to anti-Prelog through a single point mutation.13 Yu group and Xu group switched the stereoselectivity of PpYSDR and RCR from Prelog to anti-Prelog by structure-guided mutation.14,15 In our recent work, we also engineered CgKR1 into an anti-Prelog reductase toward α-halo ketones.16 Notably, these studies usually focus on a single ketoreductase and the sequences of these ketoreductases are different. Furthermore, the selected positions for mutation in the binding pocket of each ketoreductase are also different. Therefore, it’s not easy to transfer the knowledge from an engineered anti-Prelog


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ketoreductase to a Prelog ketoreductase that needs to be engineered. As such, finding a transferable solution that could switch the stereoselectivity of ketoreductase between Prelog and anti-Prelog is highly desired. The short-chain dehydrogenases/reductases (SDRs) superfamily comprises a large group of enzymes from all kingdoms, in which, several members exhibit the activities of ketone reduction.17-19 The classical enzymes in this family usually have chain length of about 250 amino acid residues, while their sequences are highly diverse. In this research, we identified the key positions that could control the stereopreferences in the sequences of several SDR enzymes, using mined LfSDR1 (from Lactobacillus fermentum) as a starting reductase. Protein engineering at related positions of LfSDR1 could switch its stereopreferences between Prelog and anti-Prelog in reduction of α-halogen-substituted acetophenones (Figure 1, 1a and 2a). Furthermore, the obtained knowledge can be transferred to other five SDR enzymes (four mined enzymes and one reported enzyme) that have 21-48% sequence identities with LfSDR1. Through mutagenesis at corresponding positions in these enzymes, their stereoselectivity for asymmetric reduction of 2-chloroacetophenone (1a) and 2-chloro-4’-fluoroacetophenone (2a) could also be switched between Prelog and anti-Prelog (Figure 1). Additionally, further mutation of these switched SDRs could access to both Prelog and anti-Prelog reduction of several aromatic ketones (3a-12a) with excellent stereoselectivity (up to >99%).


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ACS Catalysis

Figure 1. Asymmetric reduction of ketones 1a-12a following Prelog’s rule or anti-Prelog’s rule. In this model, if the hydride from the coenzyme (NADH/NADPH) attack the carbonyl of substrates (from behind the page), the different orientations of substrates in the binding pockets of enzymes will give products with opposite configurations.

RESULTS AND DISCUSSION Since ketoreductases following anti-Prelog’s rule are rare in nature, to understand better and to discovery new anti-Prelog SDR enzymes, we first performed genome mining in NCBI database using LbADH,11 a well-known anti-Prelog preference alcohol dehydrogenase from Lactobacillus brevis, as a template. From the genome of Lactobacillus fermentum, a SDR enzyme (LfSDR1) that have 47% sequence identity with LbADH was obtained (Table S2 and Figure S1 in Supporting Information). After soluble expression in Escherichia coli, reductive activities toward


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2-chloroacetophenone (1a) and 2-chloro-4’-fluoroacetophenone (2a) by LfSDR1 were tested. The α-halogen-substituted acetophenones were selected as substrates because the SDRs enzymes usually show high activity toward these compounds and the resultant halohydrins are important building blocks. As shown in Table 1, medium stereoselectivity was found in the reduction of 1a and 2a by LfSDR1, which gave the S products with 64.4% ee value and 65.2% ee value, respectively. This result revealed that the mined LfSDR1 displayed anti-Prelog stereopreference, which was same as the template enzyme LbADH. Table 1. Asymmetric reduction of ketones 1a-2a byLfSDR1 and its variants SDR

Sequences of LfSDR1

1a

2a

25.2a, 64.4b (S)c

5.0, 65.2 (S)

V186F/ 92G

>99.9, 99.9 (S)

40.3, 96.6 (S)

V186Y/ 92G

>99.9, 88.4 (S)

28.5, 98.2 (S)

V186W/ 92G

>99.9, 90.4 (S)

42.9, 99.9 (S)

V186A/G92L

86.9, 73.0 (R)

57.1, 74.3 (R)

V186A/G92L/E141L

>99.9, 95.7 (R)

60.9, 89.5 (R)

V186A/G92F/E141L

>99.9, 97.2 (R)

>99.9, 93.6 (R)

V186A/G92E/E141L

>99.9, 99.9 (R)

>99.9, 99.9 (R)

186V/ 92G (WT)

LfSDR1

a

Conversion [%]. bThe enantiomeric excess (ee) values of resultant alcohols [%], which were

measured by chiral HPLC analysis. cThe absolute configurations of resultant alcohols, which were identified by comparing the retention times of chiral HPLC with authentic samples or literature data (Table S4 in Supporting Information). The absolute configurations of resultant alcohols from Prelog reduction were shown in green, while resultant alcohols from anti-Prelog reduction were shown in blue.


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ACS Catalysis

Because LfSDR1 displayed rare anti-Prelog stereopreference, to shed light on the origins of its stereopreference and to find key residues that might control its stereoselectivity, we analyzed the structural information of LfSDR1 with reported Prelog/anti-Prolog enzymes that have similar sequences and performed multiple-sequence alignment of these enzymes. First, based on the substrate co-crystal structure of LbADH (PDB: 1ZK4),11 the structure of LfSDR1 with a closed conformation was modeled. Since LfSDR1 has high sequence identity (47%) with LbADH and displays same anti-Prelog stereopreference as LbADH, we expect the modeled structure to be useful and sufficient for next analyses. Then 2-chloro-acetophenone (1a) was docked into the structure of LfSDR1 using the AutoDock Vina program20 and the modeled substrate-enzyme complex was analyzed. As shown in Figure 2a, the chloromethyl group of substrate 1a is located in a small binding pocket (pocket A) created by V186, E141, D146, V149, and F202, while the phenyl group of 1a is surrounded by a big binding pocket (pocket B) created by residues G92 and L191. This productive orientation would lead to the production of (S)-2-chloro-1phenylethanol ((S)-1b) by following anti-Prelog’s rule, which is consistent with the experimental results of LfSDR1. In addition, substrate binding pocket A and pocket B that consisted with similar residues were also found in the structure of LbADH (Figure S4 in Supporting Information). Then multiple-sequence alignment of LfSDR1 with eight reported anti-Prelog enzymes (LbADH,11 LkADH from Lactobacillus kefir,12 LcADH from Lactobacillus composti,21 ChKRED20 from Chryseobacterium sp.,22 EbSDR8 from Empedobacter brevis,23 HPED from Aromatoleum aromaticum,24 CpSCR from Candida parapsilosis25, ScCR from Streptomyces coelicolor26) and two reported Prelog enzymes (PED from Azoarcus sp.27 and SmSDR from Serratia Marcescens28) were performed. Based on structural information analyses and multiple-


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sequence alignment (Figure S1 in Supporting Information), several positions in these enzymes were identified as key residues that might control their stereopreferences in each enzyme. As shown in Table 2, five residues (positions A1-A5) were selected in binding pocket A, while two residues (positions B1-B2) were chosen in binding pocket B. By carefully comparison of these residues, we found that the most noticeable differences between anti-Prelog and Prelog enzymes were residue A1 in pocket A and residue B1 in pocket B. For residues A1 that located in the flexible loop βF/αFG1, all reported anti-Prelog enzymes except LfSDR1 have bulky Tyr or Phe residues, while all the Prelog enzymes have smaller residues (Table 2, Figure 2b-d). For residues B1 located in loop βD/αE, all anti-Prelog enzymes have small residues, such as Ala, Gly, Ser or Pro, while Prelog enzymes have bulky residues, such as Phe or Tyr (Table 2, Figure 2b-d).


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ACS Catalysis

Figure 2. Structural analyses of modeled LfSDR1 and reported anti-Prelog/Prelog enzymes. (a) Docking of 1a into the active site of LfSDR1 model. The substrate 1a is shown in yellow sticks and spheres. The catalytic residues (S139, Y152, and K156) and NADPH are shown as white sticks. Residues that create small and large binding pockets are shown as blue sticks. The loops βD/αE and βF/αFG1 are shown in green and magenta, respectively. (b) Substrate binding pockets of LfSDR1. V186 (position A1) and F202 (position A5) in the small pocket A are shown in magenta, while G92 (position B1) in the large binding pocket B is shown in green. (c) Substrate binding pockets of anti-Prelog enzyme LbADH (PDB: 1ZK4). Y190 (position A1) in the small pocket A and A94 (position B1) in the large binding pocket B are shown in magenta and green, respectively. (d) Substrate binding pockets of Prelog enzyme PED (PDB: 2EWM). L186 (position A1) in the big pocket A and Y93 (position B1) in the small binding pocket B are shown in magenta and green, respectively. (e) Simple models of Prelog enzymes (top) and anti-Prelog enzymes (bottom). Small residues at


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position A1 of pocket A and medium to bulky residues at position B1 of pocket B might lead to Prelog enzymes. On the other hand, bulky residues at position A1 of pocket A and small residues at position B1 of pocket B probably generate anti-Prelog enzymes.

Table 2. Key residues in binding pocket A and pocket B of anti-Prelog (top) /Prelog enzymes (bottom) SDRs

A1

A2

A3

A4

A5

B1

B2

LbADH

Y190

E145

D150

L153

LkADH

Y190

E145

D150

LcADH

Y189

E144

ChKRED20

Y188

EbSDR8

References

M206

A94

L195

Ref. 11, PDB: 1ZK4

L153

M206

A94

L195

Ref. 12, PDB: 4RF2

D149

L152

M205

A93

M194

Ref. 21

H145

A150

S153

L205

G94

L193

Ref. 22, PDB: 5X8H

Y188

H145

A150

S153

E204

G94

L193

Ref. 23

HPED

F187

A144

I149

I152

L204

S93

L192

Ref. 24, PDB: 4URE

CpSCR

Y218

S174

V180

L183

K234

P122

I223

Ref. 25

ScCR

F202

L159

F164

S167

L219

G107

L207

Ref. 26, PDB: 5H5X, this study

LfSDR1

V186

E141

D146

V149

F202

G92

L191

This study

BmSDR11

F190

S147

G152

I155

A208

S95

L195

This study

PED

L186

T143

I148

Y151

NAa

Y93

T191

Ref. 27, PDB: 2EWM

SmSDR

P190

N146

F152

A155

NA

F98

M195

Ref. 28

LkSDR1

A184

A141

T146

S149

F202

T91

M189

This study

BmSDR5

A187

D144

S149

T152

Y212

T92

M192

This study


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ACS Catalysis

BsSDR4

P222

T179

N184

L187

E238

Q130

L227

This study

BsSDR8

S184

S141

F146

L149

NA

A91

I189

This study

BsSDR11

V181

A136

Y141

W144

NA

V84

M186

This study

BsSDR13

M184

L141

L146

Y149

I204

Q89

L189

This study

a

NA, not available from multiple-sequence alignment.


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In addition, further multiple-sequence alignment of LfSDR1 with a thousand homologues revealed that the residues at position A1 or position B1 exhibited very low conservation (Figure 3). Since residues at position A1 and position B1 directly extend their side chains into substrate binding pockets, their low conservation suggested that these residues might be responsible for the recognition of natural substrates with different structures. As shown in Figure 2c, in the highly simplified structure of anti-Prelog enzymes (such as LbADH), the bulky Y or F residues at position A1 might shrink pocket A while the smaller residues A or G at position B1 might enlarge pocket B, which will encourage the location of chloromethyl group of 1a in pocket A and phenyl group of 1a in pocket B. In the case of LfSDR1, the F202 at position A5 might execute similar function as the bulky residue at position A1 of other anti-Prelog enzymes (Figure 2b). On the other hand, the smaller or medium residues at position A1 of Prelog enzymes might enlarge pocket A, which will promote the location of phenyl group of 1a, while the medium or bulky residues at position B1 could prevent the binding of the phenyl group of 1a in pocket B. For example, the pocket B of Prelog enzyme PED is sterically hindered by the bulky Y93 residue at position B1 (Figure 2d), disfavoring the orientation of phenyl group of substrate in this region of the active site.27 These evidence suggest that the substrates will locate in the pockets of Prelog enzymes (such as PED, Figure 2d) with flipped configurations compared with that of anti-Prelog enzymes (such as LbADH, Figure 2c). Thus, if the phenyl group of 1a is positioned in binding pocket A (Figure 2e, top), the reduction will display Prelog stereopreference. If the phenyl group is located in binding pocket B (Figure 2e, bottom), this reductase will be an anti-Prelog enzyme such as LbADH and LfSDR1. Based on these observations, we hypothesized that protein engineering at positions A1 and B1 could reshape binding pockets A and B, which might switch


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ACS Catalysis

the stereopreferences between Prelog and anti-Prelog in SDRs through flipping the orientations of substrates in reshaped binding pockets (Figure 2e and Figure 1).

Figure 3. The conservation of residues at position A1 (top) and position B1 (bottom) in a thousand homologues of LfSDR1. The residues at position A1 (V186 for LfSDR1, red arrow) are placed after a conserved PG motif, while residues at position B1 (G92 for LfSDR1, red arrow) are also located after a conserved NNAG motif. In addition, conservations of the residues at position A1 or position B1 are very low. The homologues of LfSDR1 were searched using Phyre2 tool29 and the conservative degree of residues was determined by WebLogo 3.0 tool.30 The distributions of 20 amino acids at position A1 and B1 were also analyzed (right). To prove our hypothesis, single or double mutation at position A1 and B1 were then constructed in LfSDR1. The medium Val186 at position A1 of LfSDR1 was first mutated to bulky Phe, Tyr or Trp, intended to further reduce the space of small binding pocket A, which might improve the medium stereoselectivity of LfSDR1 by blocking the location phenyl groups of 1a in this pocket. These mutants were constructed using Overlap PCR method31 and then tested with substrate 1a and 2a using the LfSDR1 WT as a control under the same reaction conditions. Consistent with our hypothesis, the asymmetric reduction of 1a and 2a by LfSDR1


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V186F variant gave (R)-1b and (R)-2b as products, while the anti-Prelog stereoselectivity were improved to excellent 99.9% and 96.6%, respectively (Table 1). Furthermore, the LfSDR1 V186Y/W variants also displayed high stereoselectivity toward 1a and 2a (Table 1). These results indicated that shrinking binding pocket A through introducing bigger F/Y/W substitutions at V186 (Figure 4a) could improve the anti-Prelog stereopreference of LfSDR1.

Figure 4. Docking of 1a into the binding pockets of LfSDR1 V186F variant and V186A/G92L variant. (a) Shrinking small pocket A by V186F substitution (magenta) could improve the antiPrelog stereopreference of LfSDR1. (b) Pocket A was enlarged by V186A substitution (magenta) to accommodate phenyl group of 1a, while pocket B was reduced by G92L substitution (green) to block the location phenyl group of 1a, which would lead to a flipped orientation of 1a in binding pockets. This configuration of 1a in LfSDR1 V186A/G92L variant would result in Prelog alcohol as the product. Since LfSDR1 have 47% sequence identity with anti-Prelog enzyme LbADH (Table S2), it’s not surprise to improve its anti-Prelog stereopreference. Therefore, we move our attention on how to switch the stereopreference of LfSDR1 from anti-Prelog to Prelog. As previously proposed, if the phenyl group of 1a is located in binding pocket A, the reduction of 1a by


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ACS Catalysis

LfSDR1 will display Prelog-preference (Figure 2e). Thus, Val186 at position A1 of LfSDR1 were mutated to smaller Ala to enlarge pocket A for accommodation of substrates phenyl groups. Besides that, Gly92 at position B1 were substituted by medium Leu to block the location phenyl groups in pocket B. Fortunately, as shown in Table 1, the V186A/F92L variant displayed 73.0% and 74.3% stereoselectivity toward 1a and 2a. Moreover, this variant exhibited an inverse R preference while the WT exhibited an S preference. These results revealed that the substrate 1a or 2a is now located with a flipped conformation in the binding pockets created by V186A/G92L substitutions (Figure 4b, benzyl group in pocket A and chloromethyl group in pocket B), which promotes the switched Prelog selectivity, whereas the binding pockets A created by V186 in LfSDR1 WT, especially V186F/Y/W substitutions in LfSDR1 variants, were narrow and only suitable for the chloromethyl group of 1a/2a to produce the anti-Prelog stereopreference. Although the anti-Prelog and Prelog stereopreferences for asymmetric reduction of 1a/2a by LfSDR1 could be switched, the stereoselectivity of Prelog reduction by V186A/G92L variant were not high enough. To test the potential of improvement, we performed further mutagenesis using LfSDR1 V186A/G92L variant as the parent. We noticed that there is an acidic residue Glu141 at position A2 of binding pocket A (Figure 2a). The acidic side chain of Glu141 could disfavor the orientation of benzyl group of 1a in this region, which might decrease the R stereoselectivity. Therefore, Glu141 was mutated to Leu to create a hydrophobic environment and a π-alkyl interaction (Figure S5 in Supporting Information) for the accommodation of benzyl group. As shown in Table 1, improved stereoselectivity were found in the Prelog reduction of 1a and 2a by V186A/G92L/E141L variant, which gave corresponding R products with 95.7% ee value and 89.5% ee value, respectively. Besides that, we made another two mutations at position B1 of V186A/G92L/E141L variant, intended either to further shrink binding pocket A (G92F) or


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create a hydrophilic environment (G92E) in pocket A, both blocking the location of benzyl group of substrates. As shown in Table 1, V186A/G92F/E141L and V186A/G92E/E141L variants of LfSDR1 displayed more Prelog stereoselectivity toward 1a and 2a in comparison to the V186A/G92L and V186A/G92L/E141L variants. These evidences revealed that reconstruction of hydrophobic or hydrophilic environment in the binding pockets could contribute to the stereoselectivity of LfSDR1. Next, we explored the substrate scope of obtained anti-Prelog and Prelog variants of LfSDR1. Two α-halogen-substituted acetophenones (3a-4a) and eight para-/meta-/ortho-halogensubstituted acetophenones (5a-12a) were tested as LfSDR1 WT. As show in Table 3, although LfSDR1 WT exhibited medium anti-Prelog or Prelog stereoselectivity toward 3a-12a, asymmetric reduction toward most of these substrates by anti-Prelog variants and Prelog variants gave corresponding anti-Prelog products and Prelog products with high ee values (up to >99%). These results indicated that stereo-complementary reduction of 3a-12a were achieved through obtained LfSDR1 variants. Notably, some of the resultant alcohols are valuable building blocks for

construction

of

pharmaceutical

agents,

such

as

the

(R)-1-(3,5-

bis(trifluoromethyl)phenyl)ethan-1-ol ((R)-5b) for synthesis of Aprepitant.32 In addition, kinetic studies with LfSDR1 WT and some variants toward substrates 2a were also carried out to investigate the importance of positions A1/A2/B1 substitutions for the activity. As shown in Table 4, the anti-Prelog variant V186W and Prelog variant V186A/G92E/E141L exhibited corresponding 5-fold and 650-fold enhanced kcat/Km values in comparison to the WT enzyme. These results suggested that both stereoselectivity and activity for asymmetric reduction of halogen-substituted acetophenones are manipulated by the substitutions at positions A1/A2/B1 of LfSDR1. Interestingly, the Prelog variants of LfSDR1 displayed remarkable enhanced catalytic


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activity, probably due to the stabilization of the substrates in binding pocket by the π-alkyl interaction between the aromatic ring of substrates and the side chain of introduced Leu residue (Figure S5 in Supporting Information).


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Table 3. Asymmetric reduction of ketones 3a-12a by LfSDR1 WT and its variants.

SDR

Sequences of LfSDR1

3a

4a

5a

6a

7a

>99.9, 82.4 (S)

>99.9, 78.3 (S)

42.2, 86.6 (S)

8.7, 10.0 (S)

49.2, 49.5 (S)

V186F/ 92G

>99.9, 95.5 (S)

>99.9, 88.4 (S)

74.5, 72.0 (R)

14.6, 79.9 (R)

92.5, 80.6 (R)

V186Y/ 92G

>99.9, 95.7 (S)

>99.9, 99.0 (S)

60.0, 97.4 (R)

19.8, 96.6 (R)

38.1, 90.7 (R)

V186W/ 92G

>99.9, 96.6 (S)

>99.9, 99.1 (S)

44.7, 98.3 (R)

69.4, 99.5 (R)

80.7, 99.9 (R)

V186A/G92F/E141L

>99.9, 64.9 (R)

>99.9, 30.5 (R)

95.4, 94.4 (S)

19.3, 98.6 (S)

88.3, 97.8 (S)

V186A/G92E/E141L

>99.9, 99.9 (R)

>99.9, 86.5 (R)

80.9, 98.5 (S)

80.4, 95.3 (S)

68.9, 89.5 (S)

8a

9a

10a

11a

12a

13.9, 47.9 (S)

30.0, 13.9 (R)

46.0, 46.2 (R)

56.9, 46.3 (R)

>99.9, 37.2 (S)

V186F/ 92G

51.4, 71.4 (R)

81.2, 90.4 (R)

76.6, 91.3 (R)

65.9, 83.9 (R)

70.7, 42.0 (R)

V186Y/ 92G

50.3, 88.2 (R)

74.1, 97.1 (R)

71.2, 94.8 (R)

65.9, 84.6 (R)

>99.9, 90.6 (R)

V186W/ 92G

76.1, 98.3 (R)

97.0, 99.9 (R)

90.1, 99.0 (R)

84.3, 89.8 (R)

>99.9, 94,8 (R)

V186A/G92F/E141L

65.3, 85.4 (S)

99.3, 80.9 (S)

84.4, 83.3 (S)

80.4, 81.2 (S)

>99.9, 91.6 (S)

V186A/G92E/E141L

63.6, 90.6 (S)

99.4, 84.3 (S)

87.3, 87.4 (S)

85.1, 91.7 (S)

>99.9, 95.5 (S)

186V/ 92G (WT)

LfSDR1

SDR

Sequences of LfSDR1 186V/ 92G (WT)

LfSDR1


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Table 4. Kinetic parameters of LfSDR1 WT and its variants toward substrate 2a

Enzymes

Km (mM)

kcat (s-1)

kcat/Km (mM-1 s-1)

WT

4.01 ± 0.26

4.27 ± 0.25

1.06

V186F

1.97 ± 0.08

6.66 ± 0.19

3.38

V186W

1.97 ± 0.10

9.89 ± 0.41

5.02

V186A/G92F/E141L

0.41 ± 0.02

285.93 ± 5.37

697.39

V186A/G92E/E141L

0.52 ± 0.03

52.83 ± 1.17

101.59

With observations of the key residues that could control the stereoselectivity of LfSDR1 in hand, we next turned to broaden the generality of our approach in the other SDR enzymes. To identify new and diverse SDR enzymes, we performed genome mining in Bacillus megaterium, Bacillus subtilis, and Lactobacillus kefir. By using the anti-Prelog preference LfSDR1 or the Prelog preference SmSDR28 as the templates in Blast search, seven unique SDRs were mined. Besides that, a reported enzyme ScCR (PDB: 5H5X)26 was also selected as a target. The sequences of eight SDRs were added to previous sequence alignment of reported antiPrelog/Prelog SDRs. As shown in Table S2 and Figure S1 (Supporting Information), the eight SDRs have 21%-48% and 23%-35% sequence identities with LfSDR1 and SmSDR, respectively. In addition, related residues at positions A1-A5 and B1-B2 that might be important for their stereopreferences were shown in Table 2. For ScCR and BmSDR11, corresponding residues at position A1 were bulky F202 and F190, while residues at position B1 were small G107 and S95. According to our previous analyses, these compositions in binding pockets of ScCR and BmSDR11 will generate anti-Prelog SDRs. As for LkSDR1, BmSDR5, BsSDR4, BsSDR8, BsSDR11, and BsSDR13, corresponding residues at position A1 were small to medium A184, A187, P222, S184, V181, and M184, while residues at position B1 were small to medium T91,


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T92, Q130, A91, V84, and Q89 (Table 2). According to previous analyses, these compositions will generate Prelog SDRs. After soluble expression in E. coli, the activity assay of these SDRs were also carried out using 1a and 2a as substrates. As shown in Table 5, ScCR and BmSDR11 catalyzed the asymmetric reduction of 1a/2a following anti-Prelog’s rule, yielding (S)-1b and (S)-2b with 26.1%-89.0% ee values, which is consistent with our hypothesis. In the case of LkSDR1, BmSDR5, BsSDR4, BsSDR8, BsSDR11, and BsSDR13, most of them catalyzed the reduction of 1a/2a following Prelog’s rule with medium to high stereoselectivity (58.6%-99.9%), except BsSDR8 gave (S)-2b with low anti-Prelog selectivity. Table 5. Asymmetric reduction of ketones 1a-2a by SDRs and their variants.

SDRs

Sequences of SDRs

1a

2a

186V/ 92G (WT)

25.2, 64.4 (S)

5.0, 65.2 (S)

V186F/ 92G

>99.9, 99.9 (S)

40.3, 96.6 (S)

V186A/G92L

86.9, 73.0 (R)

57.1, 74.3 (R)

V186A/G92E/E141L

>99.9, 99.9 (R)

>99.9, 99.9 (R)

184A/ 91T (WT)

92.5, 97.5 (R)

>99.9, 95.1 (R)

184A/T91L/A141L

71.6, 99.3 (R)

>99.9, 99.9 (R)

A184Y/T91A

>99.9, 2.7 (S)

>99.9, 6.2 (S)

A184Y/T91G/A141E

16.7, 69.2 (S)

>99.9, 75.2 (S)

202F/ 107G (WT)

11.1, 26.1 (S)

>99.9, 87.7 (S)

5.0, 80.7 (R)

>99.9, 95.6 (R)

99.5, 58.6 (R)

99.6, 83.2 (R)

96.1, 93.4 (S)

96.1, 76.1 (S)

98.8, 88.9 (S)

99.8, 89.0 (S)

27.6, 84.7 (R)

35.2, 71.3 (R)

99.0, 99.9 (R)

99.6, 98.6 (R)

LfSDR1

LkSDR1

ScCR F202A/G107L 187A/ 92T (WT)

BmSDR5 A187F/ 92T 190F/ 95S (WT)

BmSDR11 F190A/ 95S

BsSDR4

222P/ 130Q (WT)


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NAa

NA

94.3, 62.4 (R)

95.8, 7.4 (S)

S184F/ 91A

65.2, 38.0 (R)

88.2, 0.5 (R)

S184Y/ 91A

97.9, 51.2 (R)

99.0, 3.7 (R)

S184W/ 91A

97.8, 77.6 (R)

95.6, 39.2 (R)

99.6, 99.1 (R)

59.3, 99.3 (R)

NA

NA

62.8, 99.8 (R)

99.8, 99.9 (R)

77.5, 75.6 (S)

99.0, 80.0 (S)

P222F/Q130A 184S/ 91A (WT)

BsSDR8

181V/ 84V (WT)

BsSDR11 V181F/V84A 184M/ 89Q (WT)

BsSDR13 M184F/Q89A a

NA, no measurable activity.

To transfer the knowledge that observed from engineering of LfSDR1 to our mined SDRs, similar single or double mutation at positions A1 and B1 were constructed in these enzymes. For the anti-Prelog enzyme ScCR, the bulky Phe202 residue at position A1 was mutated to smaller Ala while small residue Gly107 at position B1 was mutated to medium Leu, which might enlarge binding pocket A and shrink binding pocket B as LfSDR1 V186A/G92L variant that exhibited Prelog stereopreference. For another anti-Prelog enzyme BmSDR11, only bulky Phe190 residue at position A1 was mutated to Ala. In contrast, for the Prelog enzymes BmSDR5 and BsSDR8, their small Ala187 and Ser184 at position A1 were mutated to bigger Phe, which might create reduced binding pocket A and extrude phenyl groups of substrates out to pocket B as LfSDR1 V186F variant that displayed anti-Prelog stereopreference. In the case of the other Prelog enzymes LkSDR1, BsSDR4, BsSDR11 and BsSDR13, their small residues Ala184, Pro222, Val181, and Met184 at position A1 were mutated to bigger Phe or Tyr (LkSDR1), while the medium Thr91, Gln130, Val84, and Gln89 at position B1 were mutated to smaller A, intended to reduce binding pocket A and enlarge pocket B at the same time. These mutants were constructed


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Page 22 of 31

and then tested using substrates 1a and 2a. Consistent with our hypothesis, as shown in Table 5, the reduction of 1a and 2a by ScCR F202A/G107L variant produced (R)-1b and (R)-2b with 80.7% ee value and 95.6% ee value respectively, while the ScCR WT exhibited S preference. Although with low activities, the reduction of 1a and 2a by BmSDR11 F190A variant also gave R products. These results indicated that the stereopreferences of ScCR F202A/G107L and BmSDR11 F190A variants were switched to Prelog in comparison with the anti-Prelog stereopreferences of wild-type enzymes. In addition, the reduction of 1a and 2a by BmSDR5 A187F variant gave (S)-1b (93.4% ee) and (S)-2b (76.1% ee) as corresponding products, while reduction by BsSDR13 M184F/Q89A and LkSDR1 A184Y/T91A variants also yielded (S)alcohols as products. These results revealed that single mutation A187F of BmSDR5, double mutations M184F/Q89A of BsSDR13 and A184Y/T91A of LkSDR1 could switched their stereoselectivities from Prelog to anti-Prelog. Therefore, the stereopreferences switch either from anti-Prelog to Prelog (LfSDR1, ScCR, and BmSDR11) or from Prelog to anti-Prelog (LkSDR1, BmSDR5, and BsSDR13) were achieved through protein engineering. In addition, the Prelog and anti-Prelog stereopreferences of LkSDR1 could be further enhanced by mutations of Ala141 at position A2 to reconstruct hydrophobic or hydrophilic environment in binding pockets as LfSDR1 variants (Table 5). Although the substrate scopes of theses SDRs were not investigated, we believed that the obtained Prelog or anti-Prelog enzymes could be used as parent enzymes for further engineering toward other specific substrates. Taken together, these observations suggest the compositions and rearrangement of residues in binding pockets, especially residues at positions A1, A2, and B1, might predict and alter the stereopreferences of SDRs. However, asymmetric reduction of 1a and 2a by BsSDR8 S184F variant still exhibited Prelog stereopreference, which were same as the wild-type enzyme (Table 5). To further reduce the


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ACS Catalysis

binding pocket A that accommodates chloromethyl groups of substrates, residue at position A1 of BsSDR8 were then substituted by larger Tyr or Trp. As shown in Table 5, BsSDR8 S184Y/W variants still displayed Prelog stereopreference. In the case of BsSDR4 and BsSDR11, the P222F/Q130A and V181F/V84A variants lost their reductive activities for 1a and 2a. In order to understand the undesired results, their homologue structures were made and compared with that of LfSDR1. In the modeled structures of BsSDR4 and BsSDR11 (Figure S6 in Supporting Information), the confined space between helix αFG1 and loop βD/αE created narrower pocket B in comparison with that of LfSDR1. The smaller Ala mutations at position B1 couldn’t reshape narrow pocket B of BsSDR4 and BsSDR11, leading to the un-acceptable of substrate phenyl group that exhausted from pocket A by Phe mutations at position A1. As a result, BsSDR4 P222F/Q130A and BsSDR11 V181F/V84A variants didn’t exhibit reductive activities toward 1a and 2a. For BsSDR8, it has more than 30 amino acids at the C-terminus and also has a different structural skeleton in comparison with LfSDR1 (Figure S1 and Figure S7 in Supporting Information). This might explain why the S184F/Y/W variants didn’t switch the stereopreference of BsSDR8 WT. These observations reveal that the structural skeletons, especially helix αFG and loop βD/αE that create pocket B, are important for stereopreferences switch. Therefore, for the selection of other engineering targets, we proposed that the full sequences of SDRs should be around 250 amino acid residues and the length of helix αFG1 should be 6-7 amino acids. Because if the full lengths were too long, the SDR enzymes might exhibit atypical structural skeletons (such as BsSDR8). On the other hand, in the reported structures of anti-Prelog enzymes, the lengths of their helix αFG1 were all around 6-7 amino acids (Figure S3 in Supporting Information). If the length of helix αFG1 was more than 10 amino acids, this helix might move to loop βD/αE and create a confined binding pocket B (such as BsSDR11).


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Page 24 of 31

Although protein engineering at positions A1/B1 of Prelog enzymes BsSDR4, BsSDR8, and BsSDR11 didn’t switch their stereopreferences to anti-Prelog toward 1a and 2a, the stereopreferences of other six SDR enzymes (LfSDR1, LkSDR1, ScCR, BmSDR5, BmSDR11, and BsSDR13) could be successfully switched either from anti-Prelog to Prelog or from Prelog to anti-Prelog by only single or double mutagenesis at respective residues (Figure 5). Notably, BmSDR5 and BsSDR13 have low sequence identities (36% and 21%, respectively) with the starting enzyme LfSDR1, indicating the potential application of this approach in other homologues of LfSDR1. Furthermore, there are several un-identified SDRs that have bulky residues at poison A1 while small residues at position B1 (Figure 3), which might facilitate the discovery of more anti-Prelog ketoreductases from nature.

Figure 5. The stereopreferences toward 1a could be switched between Prelog and anti-Prelog in six SDRs through protein engineering at positions A1 and B1.

CONCLUSION


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Various ketoreductases have been optimized through protein engineering to alter their catalytic properties, such as substrate scope, activity, and especially selectivity. In many cases, the studies usually focus on a single enzyme in each and it is not easy to transfer the obtained knowledge to the other enzymes needed to be engineered. In this research, we discovered a switch between Prelog and anti-Prelog reduction toward halogen-substituted acetophenones (1a-2a) in six SDR enzymes (five mined enzymes and one reported enzyme). Based on structural information analyses and multiple sequence alignment of reported Prelog/anti-Prelog enzymes, the key residues at position A1 in binding pocket A and position

B1 in binding pocket B that might

control the stereopreferences were identified using mined LfSDR1 as a starting enzyme. Reshaping the substrate binding pockets by protein engineering at these positions of LfSDR1 could enable its anti-Prelog stereoselectivity to be improved or to be switched to Prelog. Furthermore, the knowledge obtained from LfSDR1 could be transferred to other five SDRs that have 21-48% sequence identities with LfSDR1. Mutagenesis at corresponding positions of these enzymes could switch their stereopreferences either from anti-Prelog to Prelog or from Prelog to anti-Prelog. In addition, the stereo-complementary reduction of several halogen-substituted acetophenones (3a-12a) were achieved via further optimized LfSDR1 variants. This strategy of stereopreferences switch through protein engineering at key positions A1/B1 to reshape binding pockets might be generally useful for engineering of other SDRs that have similar sequences with LfSDR1.

ASSOCIATED CONTENT


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Supporting Information. Experimental details, supporting tables and figures are provided. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] (B. Qin). *E-mail: [email protected] (X. Jia). *E-mail: [email protected] (S. You). Author Contributions ‡

F.Q. and B.Q. contributed equally.

Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work was financially supported by the Young Elite Scientists Sponsorship Program by CAST (2016QNRC001), National Natural Science Foundation of China (81602993), Science and Technology Research Projects from the Ministry of Education of the People’s Republic of China (213007A) and Project from the Department of Education of Liaoning Province. REFERENCES (1) Rudroff, F.; Mihovilovic, M. D.; Gröger, H.; Snajdrova, R.; Iding, H.; Bornscheuer, U. T. Opportunities and challenges for combining chemo- and biocatalysis. Nat. Catal. 2018, 1, 12-22. (2) Hughes, G.; Lewis, J. C. Introduction: Biocatalysis in industry. Chem. Rev. 2018, 118, 1-3.


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expressing a novel short-chain dehydrogenase/reductase gene from Serratia marcescens BCRC 10948. J. Biotechnol. 2014, 170, 6-9. (29) Kelley, L. A.; Mezulis, S.; Yates, C. M.; Wass, M. N.; Sternberg, M. J. E. The Phyre2 web portal for protein modeling, prediction and analysis. Nat. Protoc. 2015, 10, 845. (30) Crooks, G. E.; Hon, G.; Chandonia, J.-M.; Brenner, S. E. WebLogo: a sequence logo generator. Genome Res. 2004, 14, 1188-1190. (31) Heckman, K. L.; Pease, L. R. Gene splicing and mutagenesis by PCR-driven overlap extension. Nat. Protoc. 2007, 2, 924-932. (32) Zhao, M. M.; McNamara, J. M.; Ho, G.-J.; Emerson, K. M.; Song, Z. J.; Tschaen, D. M.; Brands, K. M. J.; Dolling, U.-H.; Grabowski, E. J. J.; Reider, P. J.; Cottrell, I. F.; Ashwood, M. S.; Bishop, B. C. Practical asymmetric synthesis of aprepitant, a potent human NK-1 receptor antagonist, via a stereoselective lewis acid-catalyzed trans acetalization reaction. J. Org. Chem. 2002, 67, 6743-6747.


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ACS Catalysis

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Discovery of a Switch Between Prelog and Anti-Prelog Reduction

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