Mechanistic Studies of a Non-heme Iron Enzyme OvoA in Ovothiol

Publication Date (Web): December 7, 2018. Copyright © 2018 American Chemical Society. Cite this:ACS Catal. XXXX, XXX, XXX-XXX ...
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Mechanistic Studies of a Non-heme Iron Enzyme OvoA in Ovothiol Biosynthesis Using a Tyrosine Analog, 2-Amino-3(4-hydroxy-3-(methoxyl) phenyl) propanoic Acid (MeOTyr) Li Chen, Nathchar Naowarojna, Bin Chen, Meiling Xu, Melissa Quill, Jiangyun Wang, Zixin Deng, Changming Zhao, and Pinghua Liu ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.8b03903 • Publication Date (Web): 07 Dec 2018 Downloaded from http://pubs.acs.org on December 7, 2018

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ACS Catalysis

Li Chen,†,‡,⊥ Nathchar Naowarojna,‡,⊥ Bin Chen,† Meiling Xu,‡ Melissa Quill,‡ Jiangyun Wang,§ Zixin Deng,† Changming Zhao,* ,†,‡ and Pinghua Liu*,‡ †

Key Laboratory of Combinatory Biosynthesis and Drug Discovery, Ministry of Education, School of Pharmaceutical Sciences, Wuhan University, Hubei 430072, People’s Republic of China ‡

§

Department of Chemistry, Boston University, 590 Commonwealth Avenue, Boston, Massachusetts, 02215, United States Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, People’s Republic of China

ABSTRACT: Ovothiols are thiol-histidines, which play important roles in protecting cells against oxidative stresses. Due to challenges faced in their chemical synthesis, biosynthesis provides an alternative option. In ovothiol biosynthesis, a non-heme iron enzyme (OvoA) catalyzes a four-electron oxidative coupling between L-His and L-Cys. There are debates in literature over whether oxidative C-S bond formation or sulfur oxidation is the first half of OvoA-catalysis. In this report, by incorporating a tyrosine analog, 2-amino-3(4-hydroxy-3-(methoxyl) phenyl) propanoic acid (MeOTyr), via an ambersuppressor method, we modulated the rate-limiting steps of OvoA-catalysis and observed an inverse deuterium KIE for [U-2H5]-His. In conjunction with the reported quantum mechanics/molecular mechanics (QM/MM) studies, our results suggest that Y417 plays redox roles in OvoA-catalysis and imply that oxidative C-S bond formation is most likely the first half of the OvoA-catalysis. KEYWORDS: ovothiol, sulfur-containing natural products, kinetic isotope effect, unnatural amino acid, biosynthesis

Over the last few decades, studies on sulfur-containing vitamins/cofactors (e.g., thiamin pyrophosphate,1, 2 biotin,1, 3-6 lipoic acid,7 molybdopterin,8 coenzyme B,9 coenzyme M10, 11) have provided the basis for a mechanistic understanding of many sulfur-transfer processes that are occurring in cells. The explosion of genomic information led to the discovery of many sulfur-containing natural product biosynthetic pathways, including ovothiol A 4 and ergothioneine 8 (Scheme 1).12-34 Under physiological conditions, ovothiol A exists predominantly in its thiolate form, and functions as a potent radical and peroxide scavenger.35, 36 Besides its roles against oxidative stress,37, 38 anti-cancer activities have also been reported for ovothiols.39 Thus far, industrial scale production for both ergothioneine and ovothiol has been proven to be challenging.16, 40-42 Therefore, biosynthesis was explored as an alternative option. In recent years, synergistic work between the Seebeck and Liu labs has led to the full elucidation of the aerobic ergothioneine and ovothiol biosynthetic pathways. 12-29 In these two pathways, a sulfur atom is transferred from L-Cys to an L-His side-chain by a combination of two reactions: an oxidative C-S bond formation (OvoA in ovothiol, Egt1/EgtB in ergothioneine biosynthesis, Scheme 1) and a reductive C-S lyase reaction (OvoB in ovothiol, and EgtE/Egt2 in ergothioneine biosynthesis, Scheme 1). In ergothioneine and

ovothiol biosynthesis, the sulfoxide synthases (OvoA, EgtB and Egt1) are mononuclear non-heme iron enzymes that catalyze an overall four-electron oxidation process using O2 as the oxidizing agent. Such a sulfur transfer strategy is distinct from all other well-characterized systems.12, 43 The crystal structure of Mycobacterium thermoresistibile EgtB was reported recently (Figure 1A).24 EgtB possesses a 3His ligand environment instead of the initially proposed 2-His1-carboxylate.17, 20 Upon mutation of an EgtB active site tyrosine (Y377) to phenylalanine, the EgtB Y377F mutant exhibits barely detectable sulfoxide synthase activity. Instead, this EgtB Y377F mutant efficiently mediates the oxidation of γglutamyl-cysteine (γ-Glu-Cys) to γ-glutamyl-cysteine sulfinic acid.27 Built upon this M. thermoresistibile EgtB crystal structure, three EgtB computational mechanistic studies were reported recently, using density function theory or quantum mechanics/molecular mechanics (QM/MM) methods.28, 29, 44 Conclusions from these reports differ in at least two aspects. First, there are two possibilities for the first half of this four-electron oxidation process: the sulfur oxidation suggested by Tian, Wei, and co-workers (pathway I, Scheme 1C)29, 44 or the oxidative C-S bond formation proposed by Faponle et al. (pathway II, Scheme 1C).28 Second, the EgtB Y377 could either function as a Lewis acid/base as proposed by Tian et al.,29, 44

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or it could be a part of a proton-coupled electron transfer (PCET) process as suggested by Faponle et al.28 In the analogous OvoA-catalytic process, based on these two EgtBmechanistic models, OvoA reaction may proceed via either pathway I (intermediate 10) or pathway II (intermediate 11 or 12, Scheme 1C). Owing to the lack of an OvoA crystal structure, we created an OvoA homology model (Figure S1), which suggested that OvoA Y417 is the counterpart of EgtB Y377 (Figure 1B). Indeed, in our recent report,14 by replacing Y417 in OvoA with 2-amino-3-(4-hydroxy-3-(methylthio) phenyl) propanoic acid (MtTyr, 14, Figure 1C) through amber-suppressor mediated unnatural amino acid incorporation method, we modulated the two OvoA activities: oxidative coupling and sulfinic acid formation (Equation 1, Figure 1D). However, because tyrosine and MtTyr 14 differ in both pKa and reduction potentials,45-48 the role of Y417 as a Lewis acid/base29, 44 or as part of redox chemistries remains obscure.28 To address this issue, we employed a tyrosine analog, 3-methoxytyrosine (MeOTyr, 15), which has almost the same pKa, and a much lower reduction potential (by almost 200 mV) relative to that of tyrosine (Figure 1C).49 In addition, the presence of two competing pathways in OvoA-catalysis (Equation 1, Figure 1D) enables the use of kinetic isotope effect (KIE) studies via two different approaches: kinetic isotope sensitive branching (Equation 2, Figure 1D) to measure isotope effect at one of the branching steps50-52 and substrate deuterium KIE on V/K (DV/K) using intermolecular competition.53-55 We found that replacing Y417 in OvoA with MeOTyr alters the rate-limiting step of the OvoA reaction and MeOTyr OvoA-catalysis displays an inverse deuterium KIE when deuterium-labeled histidine was used as the substrate. These results in conjunction with the mechanistic models (Scheme 1C) suggest that Y417 plays a redox role in OvoA-catalysis, and that the oxidative C-S bond formation precedes the sulfoxidation reaction in this fourelectron oxidation process.

MeOTyr was synthesized (Figures S2 and S3) and incorporated into OvoA to produce OvoAY417MeOTyr (MeOTyrOvoA).14 The identity of MeOTyr-OvoA was further verified by tandem mass spectrometry (Figures S4 and S5), which confirmed the successful replacement of Y417 with MeOTyr. The iron content of the purified MeOTyr-OvoA was determined to be 1.14±0.06 equivalent of iron using atomic emission spectroscopy (Figure S4).56, 57 The MeOTyr-OvoA was then characterized by oxygen consumption, 1H-NMR, and 13CNMR assays.17, 18 Based on the oxygen consumption rate measured by NeoFox oxygen electrode, the kinetic parameters for MeOTyr-OvoA were: kcat of 1.5 ± 0.1 s-1 and KM of 399 ± 36 M for L-His, KM of 175 ± 19 M for L-Cys (Figure S6). The KMs of both L-His and L-Cys of the MeOTyr OvoA variant are close to that of wild type OvoA (KM of 420 ± 31 M for L-His, and KM of 300 ± 34 M for L-Cys) reported previously,19 while kcat is 6.6-fold lower than that of wild type OvoA (kcat of 9.53 ± 0.33 s-1). In the 1H-NMR assay, the spectrum of the reaction mixture confirmed the production of sulfoxide 2 (Figure S7). When [β-13C]-Cys was used as the substrate for MeOTyr-OvoA, 1H- and 13C-NMR spectra indicated that cysteine sulfinic acid 13 comprises up to 26% of the product mixture (Figures 2, S8 and S9). In wild type OvoA, cysteine sulfinic acid 13 is observed as less than 10% of the product mixture.17 These results indicate that substitution of Y417 by MeOTyr in OvoA does not affect the identities of the products from OvoA catalysis; rather, this mutation alters the partitioning between oxidative coupling and cysteine sulfinic acid formation pathways (Equation 1 of Figure 1D). This result is quite different from the case of using hercynine as the substrate to replace histidine, where the C-S bond formation regioselectivity changes from the histidine δ-position to the -position.19

Scheme 1. Ergothioneine and ovothiol A biosyntheses. (A) Ovothiol A biosynthetic pathway. (B) Two aerobic ergothioneine biosynthetic pathways. (C) Two potential pathways for the oxidative C-S bond formation in OvoA-catalysis.

Figure 1. Key information related to the experimental design of the present study. (A) Reported EgtB•γGC•hercynine complex (PDB: 4X8D). (B) Predicted OvoA structure (Figure S1 for more detail). (C) Tyrosine-cysteine cross-linking in cysteine dioxygenase and two tyrosine analogs (MtTyr and MeOTyr). (D) Schematic representation of isotope sensitive branching method and equations for calculating kinetic isotope effects (KIEs).

The Methanococcus jannaschii tyrosyl amber suppressor tRNA (MjtRNATyrCUA)/tyrosyl-tRNA synthetase (MjTyrRS) pair has been developed to specifically recognize MeOTyr. 45

With active MeOTyr-OvoA, we then measured deuterium KIE using the kinetic isotope sensitive branching method (Figures 1D and 2). In the first reaction, unlabeled L-His and

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L-Cys were used as substrates, while [U- H5]-His and [β- C]-

Cys were the substrates for the second set of reaction (Figure 2A). These two reactions were quenched and mixed, and the ratios between the two products (2/2b and 13/13b) were quantified by mass spectrometry; the analytical results from one set of experiments are presented in Figure 2. The ratio of 2 and 2b ([P1]H/[P1]D, Figure 2B) is 0.68. In the same experiment, the ratio between 13 and 13b ([P2]H/ [P2]D, Figure 2C) is 0.81. Based on the kinetic model shown in Figure 1D, the KIE obtained for [U-2H5]-His is 0.84 (Equation 2, Figure 1D). This experiment was repeated at least four times and the average KIE was 0.86±0.03. This inverse KIE for [U-2H5]-His observed in MeOTyr-OvoA (Figure 2) differs significantly from that of wild type OvoA, in which the KIE for [U-2H5]-His is close to unity.14 Such differences imply that substitution of Y417 by MeOTyr changes the rate-determining step in OvoAcatalysis. In this experiment, [U-2H5]-His was used as the substrate and we will discuss in later sections. The observed KIE is most likely a secondary deuterium KIE. It is unlikely that α and β-positions will have significant contribution to this KIE. However, the measured KIE may be a combined effect from both - and δ-positions of the imidazole side-chain, while the δ-position might be the major contributor to the observed inverse secondary deuterium KIE.

and unlabeled L-His 1 at time point t and at time point zero, respectively, Figure 3A). To accurately measure the percent conversion f and the ratio between 1b and 1 at the zero point and time t, another form of L-His, [U-15N3]-His 1c was introduced. A known concentration of 1c was mixed with the reaction mixture at the zero point in a certain ratio and the mixture was analyzed by mass spectrometry. With the known concentration of 1c, and the measured ion intensities of 1, 1b, and 1c, the initial ratio of 1 and 1b can be calculated (Figure 3A). Similarly, at time point t, an aliquot of the reaction mixture was withdrawn and quenched by hydrochloric acid. The quenched reaction mixture was then mixed with a known amount of 1c and characterized by mass spectrometry to determine the ratio between 1 and 1b at time point t (Figure 3A).

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Figure 2. Measuring kinetic isotope sensitive branching KIE using [U-2H5]-His as the substrate. (A) Conditions used for the two reactions. (B) Mass spectrometry spectra of coupling product 2 and (C) Cysteine sulfinic acid 13. The ratios of compound 2 and 13 in the two sets of reactions (B & C) were used to calculate the KIE for k3 step at the branching point.

In oxygenases and oxidases, in the vast majority cases, O2 activation and the formation of oxidative species is the first irreversible step. Due to these reasons, it is not possible to measure isotope effect on V/K. However, the presence of branching pathway unmasks the isotope effect on V/K (DV/K, Equation 3, Figure 1D) and makes it measurable.50-55 Therefore, in OvoA-studies, besides the use of isotopically sensitive branching method to directly measure the isotope effect on one of the steps at the branching point, we also measured deuterium isotope effect on V/K to provide another line of evidence to support the KIE results obtained from isotopically sensitive branching studies. To measure DV/K, we conducted competition experiments using a 1:1 mixture of unlabeled LHis and [U-2H5]-His as the substrate (Figure 3A). In this method, three parameters need to be measured (Equation 9, Figure 3): the percent conversion (f) of the reaction at the time point t and the ratio between the heavy and light forms of the substrate of interest (Rs and R0 are the ratios of [U-2H5]-His 1b

Figure 3. Substrate V/K KIE measurement in an intermolecular competition experiment (A) Schematic representation of the experimental design and equations used for data analysis. L-His 1 and [U-2H5]- His 1b were mixed at a given ratio and L-Cys was then introduced to initiate the reaction. After the reaction was quenched at different time points (e.g., f = 0.6 – 0.8), [U-15N3]-His 1c of known concentration was then added to the reaction and the amount of remaining 1 and 1b was calculated based on their ion intensities relative to that of 1c. Using the same approach, the ratio between 1 and 1b prior to initiation of the reaction was also measured (f = 0). For the equation (Equation 9 in part A) used to calculate deuterium KIE on V/K, the meanings of these terms are: f (percent conversion), X1 (initial mole fraction of the unlabelled substrate 1), X1b (initial mole fraction of the labelled substrate 1b), R0 (initial substrate ratio between 1b and 1), Rs (residual substrate ratio between 1b and 1 at time point t). (B) Representative mass spectrometry results for one set of experiment at f = 0. (C) Representative mass spectrometry results for the same set of experiment at f = 0.704.

With the accurately measured R0, Rs, and percent conversion f, all of the needed parameters related to this competition experiment can be obtained (Equations 4 – 8, Figure 3A),53 which were then used to calculate deuterium KIE DV/K (equation 9, Figure 3A). The reaction was quenched at time points when the percent conversion f is in the range of 0.6 – 0.8 to minimize errors associated with KIE measurement.54, 55 Figure 3B and 3C are representative spectra at the zero time point and

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at the time point t (f = 70.4%) for one set of experiment. Detailed calculations for this set of experiments are shown in Figure S10. The experiment was repeated at least 4 times and the DV/K obtained was 0.94 ± 0.02, which is also an inverse deuterium KIE. If the kinetic model shown in Equation 1 of Figure 1D does represent OvoA-catalysis, the KIE measured from kinetic isotope sensitive branching (Figure 2 studies) should be correlated with KIE in the competition experiment (Figure 3 studies) using Equation 3 of Figure 1D. In equation 3, k3H/k5 is the ratio (74:26) of compound 2 to 13 in the reaction using unlabeled L-His and L-Cys. By incorporating Dk3 measured in Figure 2 studies into equation 3, the calculated deuterium KIE on V/K [D(V/K)] is 0.96  0.01 (Figure S10). The experimentally measured D(V/K) from Figure 3 studies is 0.94  0.02. There is a reasonably good agreement between these two sets of independent studies. Scheme 2. Proposed OvoA mechanistic models.

When Seebeck and co-workers first reported their discovery of the OvoA enzyme, they proposed several possible catalytic mechanism.21 Three computational EgtB mechanistic studies were reported recently.28, 29, 44 Due to the similarity between EgtB- and OvoA-catalysis, we included three OvoAmechanistic models here based on the mechanistic models proposed from EgtB-computational work (Scheme 2). For pathways IA and IB, the first half of the reaction is the oxidation of cysteine to a sulfenic acid intermediate (10, Scheme 2). Based on the computational studies from Tian et al., Fe(IV)=O species 10 may deprotonate imidazole δ-position to generate an anion 17, which nucleophilically attacks the sulfenic acid to produce sulfoxide 2. In this mechanistic model, Tian et al. also suggested an alternative pathway using the active site tyrosine as the base for a deprotonation process (species 19, Scheme 2).44 In pathway IB, Fe(IV)=O oxidizes the imidazole sidechain to produce a cation radical 18, which then combines with sulfenic acid to form the C-S bond in 19. In this mechanism, the active site tyrosine functions as a base to deprotonate 19 to produce 2, and this is the rate-limiting step in EgtBcatalysis based on the computational studies reported by Wei et al.29 In pathway II, Faponle et al. suggested that the active site tyrosine is part of a proton-coupled electron transfer process and in this mechanism, oxidative C-S bond formation to form intermediate 21 is the first half-reaction of the overall four-electron oxidation process.28 In addition, Faponle et al. suggested that the 21  22 reaction is the rate-limiting step

and inverse deuterium isotopic effect is predicated if the δhydrogen is replaced with deuterium (secondary deuterium isotope effect). Based on the above discussion of the two mechanistic models, in pathway I, the active site tyrosine residue most likely functions as a Lewis base and primary deuterium isotope effect as high as 5.7 for [δ-2H]-His has been predicted by Wei et al.29 In pathway II, however, Tyr417 is involved in redox chemistry and an inverse secondary deuterium isotope effect is predicted for [δ-2H]-His by Faponle et al.28 In this study, when a MeOTyr-OvoA variant is used as the catalyst, indeed, an inverse deuterium KIE was observed from studies using both kinetic isotope sensitive branching and intermolecular competition methods. This result differs from that of wild type OvoA,14 which has a KIE close to unity. Therefore, the replacement of Y417 with MeOTyr has led to a change of the rate-limiting step in OvoA-catalysis. MeOTyr-OvoA produces the same two products as that of wild type OvoA, which highly suggests that mechanistically, MeOTyr-OvoA behaves the same as wild type OvoA. However, the ratio of the two products (2 & 13, Scheme 2) changes when Y417 is replaced by an unnatural tyrosine. MeOTyr has a pKa that is almost the same as that of Tyr, while their reduction potentials are different by 200 mV.49 Therefore, it is tempting to assign the different reaction outcomes between MeOTyr-OvoA and wild type OvoA to the reduction potential differences between MeOTyr and Tyr. These two lines of evidences (an inverse deuterium isotope effect for [U-2H5]-His and the involvement of Y417 as part of redox chemistries in OvoA-catalysis), are both consistent with the mechanistic model in pathway II suggested by one of these three computational studies.28-29, 45 Therefore, our studies highly suggest that Tyr417 is playing some redox roles in OvoA-catalysis and our results in this study favor the pathway II model in Scheme 2. Notably, both Wei, Tian, and their co-workers commented that the pathway is sensitive to active site structure and there is a chance that EgtB and OvoA follow different mechanisms.29, 44 Therefore, additional studies of EgtB using our approaches in OvoA-studies could shed light on this possibility. Obtaining the OvoA structure will also be beneficial, and this is our ongoing investigations.

Experimental details, protein characterizations, NMR and MS spectra of reactions, and kinetic study results are available free of charge via the Internet at http://pubs.acs.org.

*E-mail: [email protected], and [email protected]

C. Z., with L. C., and N. N. conducted the biochemical studies with help from B. C., M. X., and M. Q. J. W. supervised the incorporation of unnatural amino acid. C. Z. and P. L. designed the experiments and Z. D. and P. L. supervised the project. N. N., C. Z., and P. L. wrote the manuscript. ⊥

These authors contributed equally.

This work is supported in part by grant from the National Science Foundation (CHE-1309148 to P.L), grants from National Natural Science Foundation of China (31670030 to C.Z., 31628004 to J.

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ACS Catalysis W.) and grant from China Postdoctoral Science Foundation (to L.C.). C. Z. is supported by Young Talents Program of Wuhan University.

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