Engineering of Cyclohexanone Monooxygenase for the

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Engineering of cyclohexanone monooxygenase for the enantioselective synthesis of S-omeprazole Yan Zhang, Yin-Qi Wu, Na Xu, Qian Zhao, Hui-Lei Yu, and Jian-He Xu ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.9b00224 • Publication Date (Web): 27 Feb 2019 Downloaded from http://pubs.acs.org on March 3, 2019

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ACS Sustainable Chemistry & Engineering

Manuscript ID: sc-2019-00224k

Engineering of cyclohexanone monooxygenase for the enantioselective synthesis of S-omeprazole Yan Zhang,a Yin-Qi Wu,a Na Xu,a Qian Zhao,b Hui-Lei Yu,a* Jian-He Xua aState

Key Laboratory of Bioreactor Engineering and Shanghai Collaborative

Innovation Center for Biomanufacturing, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, China; bJiangsu Key Laboratory of Chiral Drug Development, Jiangsu Aosaikang Pharmaceutical Co., Ltd., 766 Kening Road, Nanjing 211112, China;

*Corresponding

author: E mail: [email protected]

State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, China, Tel. +86-21-6425 2498; Fax. +86-21-6425 0840.

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ABSTRACT: Enzymatic asymmetric sulfoxidation using molecular oxygen as oxidant is a promising green chemistry approach to chiral sulfoxide production. Despite the broad substrate spectrum of cyclohexanone monooxygenases (CHMOs), some unnatural substrates with bulky functional groups, such as the pharmaceutically relevant omeprazole sulfide, cannot be effectively accepted by CHMOs. Herein, we describe a set of variants derived from an Acinetobacter calcoaceticus CHMO (AcCHMO), whose active sites adjacent to the substrate tunnel were altered to shift the substrate specificity from cyclohexanone monooxygenation toward omeprazole sulfide sulfoxidation. We performed homologous modeling and molecular docking to identify key residues that might affect the substrate specificity. Two libraries of residues lining the active center of AcCHMO were then constructed and screened by an effective halo-based selection method using the solubility difference between the substrate (omeprazole sulfide) and product (esomeprazole). Functional evaluation of the resultant variants showed that the substrate specificity of AcCHMO was markedly altered from the small natural substrate (cyclohexanone) toward the desired bulky substrate (omeprazole sulfide) despite the extremely poor activity detected even for the best variant, M2 (0.61 U/gprot). The crystal structure of M2 complexed with a flavin adenine dinucleotide (FAD) prosthetic group was determined, which provided insight into the altered substrate specificity. To improve the activity of enzyme M2 toward pharmaceutical precursor omeprazole sulfide, we performed both local and global protein engineering among the two CASTing libraries surrounding FAD+ and NADP+ prosthetic groups, and an error-prone PCR library of the full-length


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AcCHMO. As a result, variant M6 was obtained, giving a 50-fold higher activity than M2. This structure-guided protein engineering of AcCHMO provided a promising candidate for converting omeprazole sulfide into (S)-omeprazole using a green biocatalytic method.

KEYWORDS: Biocatalysis; Cyclohexanone monooxygenase; Substrate specificity; Protein engineering; Esomeprazole sulfoxide; Halo-based selection method



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INTRODUCTION Chiral sulfoxides have extensive and important applications, not only as chiral auxiliary reagents and chiral ligands, but also in pharmaceuticals, such as prazoles, which are proton-pump inhibitors (PPIs, for gastroesophageal reflux treatment; peptic ulcer disease treatment; even Zllinger-Ellison syndrome treatment).1 To date, the synthesis of chiral sulfoxides has mainly relied on the transition-metal-catalyzed asymmetric oxidation of sulfides,2 such as the synthesis of esomeprazole, the S-enantiomer of omeprazole, which requires large amounts of titanium catalyst and diethyl tartrate.3 Furthermore, the downstream removal of sulfones as over-oxidation byproducts requires tedious operations and has a large environmental impact. Enzymatic asymmetric sulfoxidation using molecular oxygen as the oxidant is considered a promising green chemistry approach to obtaining chiral sulfoxides. Type-I Baeyer–Villiger monooxygenases (BVMOs), which utilize O2 as the electron acceptor, FAD (flavin adenine dinucleotide) as the cofactor, and NADPH (nicotinamide adenine dinucleotide phosphate) as the electron donor, can catalyze the Baeyer–Villiger

oxidation

reaction.

Among

BVMOs,

cyclohexanone

monooxygenases (CHMOs), which are class B flavoprotein monooxygenases, are named according to their natural substrate, cyclohexanone.4,5 Apart from their role in nature, CHMOs can also oxidize over 100 non-natural substrates with excellent regio-, chemo-, or stereoselectivity, including the sulfoxidation and oxidation of nitrogen, boron, and selenium compounds, and epoxidation of alkenes.6–12 However, some non-natural substrates cannot be accepted by CHMOs, such as omeprazole


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sulfide, which is an important precursor in esomeprazole production (Scheme 1). The biooxidation of prazole sulfides has been explored by several groups, including whole-cell oxidation of rabeprazole and omeprazole sulfides using Cunninghamella echinulata MK40 and Lysinibacillus sp. B71.13,14 Where rabeprazole and omeprazole were accumulated up to 2.5 g/L and 0.115 g/L, respectively, with high enantiomeric excesses (ee) (>99%, (S)-enantiomer). Recently, we discovered two novel

Type-I

BVMOs,

BoBVMO

and

AmBVMO,

from

Bradyrhizobium

oligotrophicum and Aeromicrobium marinum, respectively, that catalyze sulfides oxidation. Notably, these two BVMOs catalyzed the sulfoxidations of bulky prazole sulfides,

including

omeprazole,

ilaprazole,

rabeprazole,

pantoprazole,

and

lansoprazole sulfides.15 However, the opposite product configuration to that desired was obtained when omeprazole sulfide was used as substrate. Furthermore, the poor thermostability and soluble expression levels limited their further application. In addition to discovering new strains or enzymes, structure-guided protein engineering is a powerful tool for extending the scope of substrate acceptance, and enhancing the thermostability and stereoselectivity of BVMOs. To gain insight into the catalytic mechanism and structure–function relationships, and provide guidance for protein engineering, many scientists have committed to solving the crystal structures of BVMOs. However, very few crystal structures of BVMOs are available at present. The first solved BVMO crystal structure was that of phenylacetone monooxygenase (PAMO), which originates from moderately thermophilic bacterium Thermobifida fusca, combined with a FAD prosthetic group in the structure.16 In



contrast to CHMOs, PAMO is a thermally robust enzyme with a relatively narrow range of substrate acceptance.17 Therefore, the solved crystal structure of PAMO has been exploited for the structure-inspired enzyme redesign of PAMO. Through fragment assembly of PAMO and CHMONCIMB9871, the two enzymes were successfully switched into phenylcyclohexanone monooxygenase (PCHMO), which resulted in broadened substrate acceptance while maintaining the thermostability.18 Subsequently, the first crystal structure of a CHMO from Rhodococcus sp. strain HI-31 (RmCHMO) was published in 2009, which provided mechanistic insight into the CHMO-catalyzed Baeyer–Villiger oxidation.19,20 As the sequence identity of RmCHMO and CHMONCIMB9871 was 57%, the solved crystal structure of RmCHMO provided an important template for the homology modeling of CHMONCIMB9871. Accordingly, other protein engineering approaches have been used to alter the substrate specificity, increase or invert the enantioselectivity, and improve the thermostability of BVMOs.21–35 These reports have inspired us to evolve BVMOs as catalysts for the oxidation of pharmaceutically relevant bulky sulfides through structure-guided protein engineering. Meanwhile, a high-throughput screening method is necessary to improve the screening efficiency of mutant libraries. The common method used to determine BVMO activity involves measuring NAD(P)H consumption, which does not suit the very low activity of BVMOs. The other methods for screening BVMO mutant libraries depend on either HPLC/GC analyses or coupling with other reactions indirectly.36–38 The engineered variants of CHMONCIMB9871 disclosed in a Codexis patent


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showed the desired enantioselectivity for oxidizing prochiral omeprazole sulfide into (S)-omeprazole.39,40 However, its structure–function relationships are unknown. Herein, we altered the substrate specificity and improved the catalytic activity of a CHMO from Acinetobacter calcoaceticus (AcCHMO, with 70% identity to CHMONCIMB9871) through semi-rational protein engineering. We divided the enzyme structure into two regions, namely, the core region, including the FAD binding sites, NADPH binding sites, and substrate tunnel, and the surface region (Fig. 1). Different mutagenesis methods were adopted for these two regions. Furthermore, the crystal structure of variant M2 was solved by X-ray diffraction at a resolution of 2.2 Å, showing that the structural changes might be responsible for the dramatically varied substrate specificity. Furthermore, we invented an effective plate assay method based on the solubility difference between the substrate (omeprazole sulfide) and product (esomeprazole sulfoxide), where the mutant activity was directly correlated to the sizes of transparent halos on Luria–Bertani (LB) agar plates.

EXPERIMENTAL SECTION Chemicals and Materials. All prazole sulfides, sulfoxides, and sulfones were provided by Aosaikang Pharmaceutical Co., Nanjing, China. All other commercial chemicals were purchased from TCI, Macklin, Aladdin, or Sigma-Aldrich. PrimerSTAR HS and rTaq polymerase, restriction enzymes (Dpn I, Nde I, and Hind III), and T4 DNA ligase were purchased from TaKaRa Biotechnology Co., Dalian, China. Primers were synthesized by Generay Biotech Co., Shanghai, China.



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Homology Modelling and Molecular Docking. Homology modelling of AcCHMO was performed using Modeller 9.15 software based on the crystal structures of FAD- and NADPH-dependent TmCHMO from Thermocrispum municipale (protein databank (PDB) ID: 5M10) or RmCHMO from Rhodococcus sp. strain HI-31 (PDB ID: 3UCL) with a sequence identity of 58%. The stereochemistry of the model was evaluated using a Ramachandran plot, and the percentage of residues in the allowed region was 97.4%. Molecular docking was performed with AutoDock Vina using the default program parameters. The center coordinates of the grid box were calculated by visual molecular dynamics (VMD), and the size of the grid box was set as 24 Å in each dimension. The docking results were selected according to their binding affinities and molecule conformations.41–43 CAVER 3.0 was used to identify the tunnels existing in AcCHMO (http://www.caver.cz).44 Mutant

Library

Construction.

Site-directed

mutagenesis

(SDM)

and

combinatorial active site saturation testing (CASTing) were introduced by PCR into the pET28a-AcCHMO template DNA (either WT or variants) using the QuikChange site-directed mutagenesis protocol.45 The PCR mixture (50 L) contained plasmid (Template, 50 ng), 1.25 U PrimerSTAR HS, and both the forward and reverse primers. The reaction mixture was preheated at 98 °C for 10 s, annealed at 55 °C for 30 s, and elongated at 72 °C for 7 min, which was repeated for 30 cycles. The PCR product was digested with 0.5 U Dpn I at 37 °C for 1 h. Plasmids containing the mutated gene were transformed into E. coli BL21(DE3) host cells and then plated on an LB agar plate with 50 g/mL kanamycin. The sequence of each variant was


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confirmed by DNA sequencing at Saiyin Biotechnology Co., Shanghai, China. Error-prone PCR (epPCR) was conducted using plasmid pET28a-M5 as the template and 50 M Mn2+ was selected for the desired mutagenesis rate (1–3 mutation sites per gene based on sequence analysis of 20 samples). The PCR product was digested with Nde I and Hind III restriction enzymes. The digested product was then ligated into the corresponding sites of pET28a. The recombinant plasmid was then transformed into E. coli BL21 (DE3) cells. The primers used in this study are listed in Table S1. Mutant Library Screening. A stepwise three-round screening strategy was adopted. Primary screening was conducted by inoculation onto LB agar plates supplemented with kanamycin (50 g/mL), isopropyl--ᴅ-thiogalactopyranoside (IPTG, 0.1 mM), omeprazole sulfide (2 mM), and cosolvent DMSO (1%, v/v). After incubation at 30 °C for 12 h, the potentially positive clones with visible halos (larger than the control) produced by enzymatic monooxygenation of the less water-soluble omeprazole sulfide were selected and further cultured in LB liquid medium (4 mL) for secondary screening. The harvested cells were resuspended in 100 mM potassium phosphate buffer (KPB, pH 9.0) containing 1 mM omeprazole sulfide and 1 mM NADPH (500 L total volume). After the reacting at 30 °C for 1 h, the conversion and ee were determined by HPLC, as described previously.15 The specific activity of positive variants with enhanced conversion rates was further measured using purified enzymes as the third round of screening.



Protein Expression and Purification. The E. coli cells expressing wild-type and variants of AcCHMO were cultivated in LB medium at 37 °C and 180 rpm. When the optical density at 600 nm (OD600) reached 0.6–0.8, IPTG was added to a final concentration of 0.2 mM and the cultivation temperature was decreased to 16 °C for protein overexpression. After cultivation for 12 h, the cells were harvested by centrifuging at 5,000 g for 10 min, and the pellets were washed twice with ice-cooled 100 mM KPB (pH 9.0). The resultant cells were resuspended in the same buffer and disrupted by ultrasonication. The cell lysates were centrifuged at 12,000 g at 4 °C for 30 min to remove the particulate fraction. The supernatant was loaded onto a HisTrap Ni-NTA FF column (5 mL, GE Healthcare) pre-equilibrated with buffer A (50 mM KPB, 500 mM NaCl, 10 mM imidazole, pH 8.0). The protein was eluted using an increasing gradient of imidazole, from 10 to 150 mM, at a flow rate of 5 mL/min. The resulting pure protein was collected and concentrated by ultrafiltration. The purified proteins from Ni2+ affinity chromatography were further polished through gel-filtration chromatography (SuperoseTM 12 10/300 GL, GE Healthcare) (Fig. S1). The fractions were determined by SDS-PAGE (Fig. S2). The peak fractions were concentrated to 14 mg/mL and used for crystallization. Crystallization and Diffraction Data Collection. Crystals were grown at 18 °C using the sitting-drop vapor diffusion method 46 by mixing protein solution (2 L, 14 mg/mL) with reservoir solution (2 L) containing 25% (w/v) PEG 3350, 0.1 M Tris-HCl (pH 8.5), and 0.2 M NaCl. Crystals were cryoprotected with reservoir solution plus 15% glycerol. X-ray diffraction data were collected using beamline


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BL19U2

47

at the National Center for Protein Science (Shanghai, China). Native and

derivative datasets were processed using XDS

48

and HKL3000 software

49,

respectively. Structure Determination and Refinement. The initial diffraction phases were determined by a molecular replacement method using the crystal structure of RmCHMO from Rhodococcus sp. HI-31 (PDB ID: 3UCL) as a search model. Phaser software 50 was used for single-wavelength anomalous diffraction (SAD) phasing and PHENIX. Autobuild

51

was used to build the initial model. The initial model and

phase information were then transferred to the native data set. Other parts of the structure were manually built in COOT

52

and iteratively refined using

PHENIX.Refine.53 In the final model, more than 97.0% of residues fell in the favored region in the Ramachandran plot and the final Rwork/Rfree values were 0.1848/0.2290, as shown in Table S4. The PDB coordinate was deposited with entry ID 6A37. Specific Activity and Enantioselectivity Assays. The specific activity and stereoselectivity of AcCHMO variants toward cyclohexanone (1a), thioanisole (1b), 5-methoxy-2-methylthio-1H-benzimidazole (1c), and omeprazole sulfide (1d) was measured using purified enzymes. In a 500-L reaction system, 2 mM substrate 1a or 1b (0.2 mM for 1c or 1d), 2 mM NADPH for 1a or 1b (0.2 mM NADPH for 1c or 1d), and diluted enzyme were mixed in KPB (100 mM, pH 9.0). After the reaction was performed at 30 °C for 10 min with mixing at 1000 rpm in a Thermomixer (Eppendorf, Germany), the reaction mixture was extracted with an equal volume of ethyl acetate. The specific activity and enantioselectivity were determined by GC or



HPLC, as described elsewhere.15 Enzymatic Sulfoxidation of Omeprazole Sulfide to Producing Esomeprazole. The 10-mL reaction mixture was composed of 3 g/L omeprazole sulfide as substrate (with 10% (v/v) DMSO), ᴅ-glucose (1.5 equiv.), purified enzyme M5 or M6 (1 g/L), glucose dehydrogenase (GDH, 2 g/L), NADP+ (0.2 mM), and potassium phosphate buffer (100 mM, pH 9.0), and was shaken at 180 rpm and 25 °C. Samples were intermittently removed and extracted to analyze the conversion rate by HPLC.

RESULTS AND DISCUSSION Construction of a Halo-based Plate Assay Method. To improve the efficiency of mutant library screening, we designed and constructed an effective halo-based selection method, according to our previous experience with an esterase assay.54 The medium components for the plate assay were first optimized in terms of substrate concentration, cosolvent ratio, and IPTG addition. As a preliminary experimental result, when 2 mM omeprazole sulfide and 1% (v/v) DMSO were added, the transparent LB agar plate turned milky white, as shown in Fig. S3. When no IPTG was added, the colonies grew well, but without any transparent halos on the plate (Fig. S3A). In contrast, when 0.1 mM IPTG was added, obvious transparent halos appeared, but the colonies grew slower (Fig. S3B). The real mutant library screening plate is shown in Fig. S3C. Focused Mutagenesis in the Core Region of AcCHMO. The residues lining the substrate binding pocket and substrate tunnel were more likely to have a profound


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effect on the enzyme activity and substrate specificity. Based on the homology modelling, molecular docking results, and hot sites in the literature, two regions of substrate binding sites, one adjacent to the substrate tunnel (Library A) and the other buried inside the protein (Library B), were selected (Fig. 2A). Two other regions were also considered (Fig. 2B), covering amino acid residues surrounding the FAD prosthetic group (Library C) and NADP+ prosthetic group (Library D). Accounting for the primary sequence and three-dimensional structure simultaneously, we attempted protein engineering based on combinatorial active-site saturation testing (CASTing) and iterative saturation mutagenesis (ISM), as proposed by Reetz et al.55– 57

Nineteen residues in libraries A and B were divided into eleven groups, considering

the synergistic effect between them. While twenty-two residues in libraries C and D were also divided into eleven groups, using the same approach. The group details are shown in Table S2, and the degeneracy NNK or NDT codons (N = A, G, C, T; K = G, T;

D

=

A,

G,

T;

NDT

involves

12

codons

for

12

amino

acids

(N/S/I/D/G/V/Y/C/F/H/R/L) and NNK involves 32 codons for 20 amino acids) were used to construct the locally focused mutagenesis libraries of AcCHMO. The mutant libraries were rapidly screened using the halo-based plate assay, as described in the Experimental Section. The screening results of mutagenesis libraries are listed in Table 1. In the first round, a double mutation variant, K326C/F432L (M1), was identified, showing new trace activity towards omeprazole sulfide. Subsequently, using M1 as the parental enzyme, we performed ISM repeatedly, resulting in an octuple mutation variant (M2) that exhibited a specific activity of 0.61



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U/gprotein toward omeprazole sulfide (Table 1). The CASTing experiments of Library C and Library D were also implemented using M2 as parent. As a result, two variants, M3 and M4, showed nearly six-fold greater omeprazole sulfide sulfoxidation activity than that of parental M2. The simple combination of M3 and M4 created a better variant, M5, which presented a near-20-fold improvement compared with M2, suggesting that a strong synergistic effect might exist between M3 and M4. Error-prone

PCR

Mutagenesis

of

Full-length

AcCHMOM5.

Random

mutagenesis can be used to effectively identify new hot sites. Therefore, error-prone PCR was employed in the second round of mutagenesis using M5 as a template to generate a random library. After screening approximately 5,000 clones and confirming the specific activity, eight variants showing specific activity improvements of more than 1.3-fold were identified (Table S3). Among them, the best variant was M6 (M5L143P/K269E), which showed 2.6-fold higher activity compared with M5 (Table 1). Although these improvements were not remarkable, many new mutation sites beneficial for activity improvements were identified in this round. Two of the mutation sites located just within the core region (L143, G185) were newly identified despite being missed in the first round of semi-rational mutagenesis. The remaining sites were situated on the surface region (Fig. S4). Furthermore, to achieve a combinatorial improvement in catalytic efficiency, we also performed DNA shuffling

58,59

using the genes found in the epPCR library. Unfortunately, no superior

variants were obtained after a screening of 3,000 colonies in total. Crystal Structure and Substrate Specificity Analysis of AcCHMO Variants. To


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elucidate the mechanism responsible for substrate specificity changes in AcCHMO variants, we attempted to solve the crystal structures of AcCHMOs WT, M1, M2, and M5. However, only the crystal structure of AcCHMOM2 complexed with an FAD prosthetic group was successfully resolved in the space group P21 at a 2.2 Å resolution using the molecular replacement method (Table S4). This structure contained two Rossmann folds and exhibited typical BVMO domain organization with an FAD domain (residues 1–141 and 387–542), an NADP domain (residues 152–210 and 334–380), and a helical domain (residues 224–333) (Fig. S5A). CHMOs are known to have two forms, namely, CHMOopen and CHMOclosed structures. The closest structures to AcCHMO were those reported for TmCHMO and RmCHMO (both with 58% sequence identity). When compared with the CHMOopen (PDB ID: 3GWF) and CHMOclosed (PDB ID: 3GWD) forms of RmCHMO, the resulting root-mean-square deviation (rmsd) values were 0.572 and 0.825 Å, respectively. This indicated that the structure of AcCHMOM2 was much closer to the “open” form, which was consistent with the real structure in a non-catalytic state. The location of the FAD prosthetic group was also consistent with that of RmCHMO (Fig. S5B). To gain insight into the origin of the dramatically altered substrate specificity, the structures of AcCHMOM2 (PDB ID: 6A37) and AcCHMOWT (modelled based on the crystal structure of AcCHMOM2) were compared. As shown in Fig. 3, four mutation sites replaced by smaller amino acids (K326C, F432L, T433A, and L435S) in AcCHMOM2 occurred at locations close to the substrate tunnel causing the substrate tunnel to significantly broaden, which was beneficial for accepting sterically bulky



substrates. The newly formed interaction between hydroxy Y246 and the sulfurous substrate aided substrate binding in the catalytic pocket. The three mutation sites (E488K, S489C, and W490R) of M4 were either surrounding the NADP+ prosthetic group or located on a flexible loop (residues 487–504), which has been demonstrated to have interactions with the NADP+ molecule.60 Here, three additional hydrogen bonds formed between the three mutation sites and NADP+, resulting in an enzyme with drastically increased activity (Fig. 4). Similarly, the dramatically improved activity of variant M3 was attributed to two additional hydrogen bonds formed between FAD and the newly created residues (Fig. 4), which were beneficial for FAD binding. Through two rounds of directed evolution, the catalytic activity of AcCHMO variants specific to pharmaceutically relevant omeprazole sulfide increased gradually, as shown in Fig. 5. The combinatorial multiple mutation variant M6 showed 5,611-fold higher activity compared with variant M1. To explore the evolutionary footprints of AcCHMO variants with dramatically altered substrate specificity, four distinct substrates with varying sizes and structures, including cyclohexanone (1a), thioanisole (1b), 5-methoxy-2-methylthio-1H-benzimidazole (1c), and omeprazole sulfide (1d), were selected to fingerprint the activities of AcCHMOWT and six variants. Interestingly, compared with the bulky omeprazole sulfide, the catalytic activity of the variants toward small substrates cyclohexanone and thioanisole decreased obviously during the evolutionary course. In particular, for native substrate cyclohexanone, variant M2 had lost its natural activity completely. However, the activity of variants


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toward substrate 1c initially increased and then decreased later. These results showed an evolutionary route of AcCHMO, demonstrating that the substrate specificity was altered stepwise from the natural small substrate (cyclohexanone) to the pharmaceutically important bulky omeprazole sulfide. Catalytic Performance of AcCHMO Variants for (S)-omeprazole Synthesis. The engineered AcCHMOs were capable of catalyzing the asymmetric sulfoxidation of prochiral omeprazole sulfide into pharmacologically active (S)-omeprazole, accompanied by the consumption of NADPH to form NADP+. Therefore, a glucose dehydrogenase from Bacillus megaterium was introduced to regenerated the desired cofactor NADPH from NADP+ using glucose as a cheap cosubstrate. The enzymatic sulfoxidation of omeprazole sulfide (3 g/L) was conducted comparatively using the best two variants, M5 and M6, using a 1 g/L dose of each purified enzyme under the same conditions. As shown in Fig. 6, the reaction catalyzed by variant M6 achieved >95% conversion after 22 h. Meanwhile, variant M5 reached only 14% conversion after 22 h. In contrast to the chemical oxidation of omeprazole sulfide using metal catalysts and peracids or hydrogen peroxide, this enzymatic process used oxygen (air) and glucose as sustainable cosubstrates, affording water as a clean byproduct, making this method a promising green chemistry approach to esomeprazole production.

CONCLUSIONS In summary, we applied structure-guided rational and random strategies for



evolving a putative cyclohexanone monooxygenase to activate its potential activity toward the stereoselective sulfoxidation of bulky omeprazole sulfide. An effective halo-based plate assay method was designed and constructed based on the solubility difference between the substrate (omeprazole sulfide) and product (esomeprazole). Compared with the common HPLC/GC assays or microtiter-based screening methods of monooxygenases, which monitor the NADPH absorbance at 340 nm, the plate assays significantly improved the screening efficiency of mutant libraries. As the solubilities of sulfoxides and sulfones are generally higher than those of the corresponding sulfides, this halo-based plate assay method could be applied extensively. Consequently, a 5,611-fold improved variant (M6) was hit after two rounds of evolution, including a combination of CASTing and ISM of the core sites and epPCR mutation of the full-length protein. The footprints on the AcCHMO evolution road were explored with migratory substrate specificity toward four different substrates with varying sizes and structures. The substrate specificity of AcCHMOs was shifted from the small and natural substrate (cyclohexanone) to the bulky and unnatural omeprazole sulfide. We also determined the crystal structure of AcCHMOM2 complexed with an FAD prosthetic group, and analyzed the structural transformation between AcCHMOWT and its variants. In the majority of flavoenzymes, the cofactor is tightly but noncovalently bound.61 However, in our crystallization experiments, even when no external FAD was added, the FAD prosthetic group was still observed in the crystal structure of AcCHMOM2. Furthermore, activity experiments showed that a less than two-fold


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increase was observed even when saturated FAD was added. This was consistent with the literature (cofactor is tightly but noncovalently bound) described above. The resulting best variant (M6) converted omeprazole sulfide into (S)-omeprazole with greatly enhanced efficiency, making it a promising candidate for practical applications. Further analysis and reaction optimization studies are ongoing to better elucidate the unknown mechanism behind the substrate specificity shifts of CHMOs and to promote their industrial application.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Table S1-S4 and Figure S1-S7. AUTHOR INFORMATION Corresponding Author H-L Yu. Email: [email protected] Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS The work was financially supported by the National Natural Science Foundation of



China (Nos. 21536004, 21672063 and 21871085) and the Fundamental Research Funds for the Central Universities (No. 22221818014). We thank Yue-Peng Shang and Feng Liu at East China University of Science and Technology for their insightful discussions. We thank Simon Partridge, PhD, from Liwen Bianji, Edanz Editing China (www.liwenbianji.cn/ac), for editing the English text of a draft of this manuscript.


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Table Captions Table 1. Properties of the purified AcCHMO variants.a

Figure Legends Scheme 1. Asymmetric oxidation of omeprazole sulfide by BVMOs for the production of S-omeprazole. Figure 1. The enzyme structure is divided into two regions for focused evolution: 1) The core region (FAD binding, NADPH binding and substrate tunnel); and 2) The surface region. CASTing and ISM were employed to mutate the residues in the core region. epPCR was further employed to discover further reinforcing mutations in the surface region. Figure 2. Poses of omeprazole sulfide docked in the modelled structure of AcCHMOWT. The target amino acid residues of Libraries A to D are shown in sticks. The substrate tunnel is shown in surface. The substrate omeprazole sulfide is shown in salmon; FAD prosthetic group in orange; and NADP+ in yellow. Libraries A to D are shown in red, green, marine and cyan, respectively. Figure 3. Structural comparison between AcCHMOWT (A) and AcCHMOM2 (B). The structure of AcCHMOWT was built based on the crystal structure of AcCHMOM2 (PDB ID: 6A37). The amino acid residues, substrate and FAD are shown in sticks. The substrate tunnel is shown in surface. Figure 4. Comparison of hydrogen bonding in AcCHMOWT (A) and its variant AcCHMOM5 (B). The amino acid residues, NADP+ and FAD are shown in sticks. The hydrogen bonds are shown in imaginary lines. Figure 5. Activity fingerprints of AcCHMOWT and its variants (M1 to M6) with different substrates. The activity was measured by GC or HPLC and the relative activity was expressed as a percentage of the maximum activity toward each substrate. Figure 6. Progress curves of omeprazole sulfide sulfoxidation catalyzed by the purified enzymes of two AcCHMO variants: M5 (♦) and M6 (■). The enzymatic sulfoxidation of omeprazole sulfide (3 g/L) was performed at pH 9.0 and 25 °C with a dose of each 1 g/L purified enzymes under the same condition. GDH was used to recycle NADPH.


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TOC Synopsis: We applied structure-guided hierarchical strategies and an effective halo-based plate assay method for evolving AcCHMO to alter its substrate specificity from the native substrate cyclohexanone to the pharmaceutically important bulky omeprazole sulfide.


ACS Sustainable Chemistry & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46

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Table 1. Properties of the purified AcCHMO variants.a Enzyme and targeted area

Mutation sites

Specific activity (U/gprotein)a

% ee (Config.)b

WT, starting enzyme

None

ndc

nd -3

1 (M1), substrate tunnel

K326C/F432Ld

(5.4 ±0.5)  10

29 (S)

2, substrate tunnel

K326F/F432L

nd

nd

3, substrate tunnel

K326C/F432I

(4.4 ±0.4)  10

4, substrate tunnel

K326C/F432L/L435S/S438I

(2.5 ±0.5)  10

5, substrate tunnel

K326C/F432L/T433C/L435S/S438I

(3.2 ±0.8)  10

6, substrate tunnel

K326C/F432L/T433A/L435S/S438I

(4.3 ±0.1)  10

7, substrate tunnel

K326C/L426F/F432L/T433A/L435S/S438I

(9.0 ±0.3)  10

-3

4.9 (S)

-2

57 (S)

-2

84 (S)

-2

83 (S)

-2

69 (S)

-2

8, substrate tunnel

K326C/L426P/F432L/T433A/L435S/S438I

(9.9 ±0.2)  10

16 (S)

9, substrate tunnel

K326C/L426F/F432L/T433A/L435S/S438I/F505L

0.12 ± 0.01

93 (S)

10 (M2), substrate tunnel

F246Y/K326C/L426F/F432L/T433A/L435S/S438I/F505L

0.61 ± 0.02

97 (S)

11 (M3), FAD binding

F246Y/K326C/L426F/F432L/T433A/L435S/S438I/F505L/N386S/I388K/M390I

3.8 ± 0.1

99 (S)

12 (M4), NADPH binding

F246Y/K326C/L426F/F432L/T433A/L435S/S438I/F505L/E488K/S489C/W490R

3.5 ± 0.1

99 (S)

12 ± 1

99 (S)

30 ± 1

99 (S)

13 (M5), simple combination

14 (M6), surface a

F246Y/K326C/L426F/F432L/T433A/L435S/S438I/F505L/N386S/I388K/M390I/E488K/S4 89C/W490R L143P/F246Y/K269E/K326C/L426F/F432L/T433A/L435S/S438I/F505L/N386S/I388K/M 390I/E488K/S489C/W490R

Specific activity was determined at pH 9.0 and 30 °C using purified enzyme and omeprazole sulfide. b ee value was determined by HPLC. c Not detected. d The bold

mutations indicate newly involved mutation(s) in each round.


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Figure 1. The enzyme structure is divided into two regions for focused evolution: 1) The core region (FAD binding, NADPH binding and substrate tunnel); and 2) The surface region. CASTing and ISM were employed to mutate the residues in the core region. epPCR was further employed to discover further reinforcing mutations in the surface region. 29x16mm (600 x 600 DPI)



Figure 2. Poses of omeprazole sulfide docked in the modelled structure of AcCHMOWT. The target amino acid residues of Libraries A to D are shown in sticks. The substrate tunnel is shown in surface. The substrate omeprazole sulfide is shown in salmon; FAD prosthetic group in orange; and NADP+ in yellow. Libraries A to D are shown in red, green, marine and cyan, respectively. 31x15mm (600 x 600 DPI)


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Figure 3. Structural comparison between AcCHMOWT (A) and AcCHMOM2 (B). The structure of AcCHMOWT was built based on the crystal structure of AcCHMOM2 (PDB ID: 6A37). The amino acid residues, substrate and FAD are shown in sticks. The substrate tunnel is shown in surface. 31x15mm (600 x 600 DPI)



Figure 4. Comparison of hydrogen bonding in AcCHMOWT (A) and its variant AcCHMOM5 (B). The amino acid residues, NADP+ and FAD are shown in sticks. The hydrogen bonds are shown in imaginary lines. 31x15mm (600 x 600 DPI)


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Figure 5. Activity fingerprints of AcCHMOWT and its variants (M1 to M6) with different substrates. The activity was measured by GC or HPLC and the relative activity was expressed as a percentage of the maximum activity toward each substrate. 15x11mm (600 x 600 DPI)



Figure 6. Progress curves of omeprazole sulfide sulfoxidation catalyzed by the purified enzymes of two AcCHMO variants: M5 (♦) and M6 (■). The enzymatic sulfoxidation of omeprazole sulfide (3 g/L) was performed at pH 9.0 and 25 °C with a dose of each 1 g/L purified enzymes under the same condition. GDH was used to recycle NADPH. 19x11mm (600 x 600 DPI)


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Asymmetric oxidation of omeprazole sulfide by BVMOs for the production of S-omeprazole. 77x16mm (600 x 600 DPI)



Synopsis: We applied structure-guided hierarchical strategies and an effective halo-based plate assay method for evolving AcCHMO to alter its substrate specificity from the native substrate cyclohexanone to the pharmaceutically important bulky omeprazole sulfide. 22x13mm (600 x 600 DPI)


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Engineering of Cyclohexanone Monooxygenase for the

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