Mechanistic Studies of a Nonheme Iron Enzyme Ovoa in Ovothiol

Letter

Cite This: ACS Catal. 2019, 9, 253−258 pubs.acs.org/acscatalysis

Mechanistic Studies of a Nonheme Iron Enzyme OvoA in Ovothiol Biosynthesis Using a Tyrosine Analogue, 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*, † 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

*S Supporting Information

ABSTRACT: Ovothiols are thiol-histidines that play important roles in protecting cells against oxidative stresses. Because of challenges faced in their chemical synthesis, biosynthesis provides an alternative option. In ovothiol biosynthesis, a nonheme iron enzyme (OvoA) catalyzes a four- electron oxidative coupling between L-His and L-Cys. There are debates in the 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 analogue, 2-amino-3-(4-hydroxy-3-(methoxyl) phenyl) propanoic acid (MeOTyr), via an amber-suppressor method, we modulated the rate-limiting steps of OvoA-catalysis and observed an inverse 2 deuterium KIE for [U- H5]-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

ver the last few decades, studies on sulfur-containing synthases (OvoA, EgtB, and Egt1) are mononuclear nonheme vitamins/cofactors (e.g., thiamin pyrophosphate,1,2 bio- iron enzymes that catalyze an overall four-electron oxidation O− 1,3 6 7 8 9 tin, lipoic acid, molybdopterin, coenzyme B, coenzyme process using O2 as the oxidizing agent. Such a sulfur transfer M10,11) have provided the basis for a mechanistic under- strategy is distinct from all other well-characterized sys- standing of many sulfur-transfer processes that are occurring in tems.12,43 Downloaded via INST OF BIOPHYSICS on March 1, 2019 at 01:29:56 (UTC). cells. The explosion of genomic information led to the The crystal structure of Mycobacterium thermoresistibile EgtB discovery of many sulfur-containing natural product biosyn- was reported recently (Figure 1A).24 EgtB possesses a 3-His thetic pathways, including ovothiol A 4 and ergothioneine 8 − ligand environment instead of the initially proposed 2-His-1- See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles. 12 34 (Scheme 1). Under physiological conditions, ovothiol A carboxylate.17,20 Upon mutation of an EgtB active site tyrosine exists predominantly in its thiolate form and functions as a (Y377) to phenylalanine, the EgtB Y377F mutant exhibits potent radical and peroxide scavenger.35,36 Besides its roles 37,38 barely detectable sulfoxide synthase activity. Instead, this EgtB against oxidative stress, anticancer activities have also been ffi γ 39 Y377F mutant e ciently mediates the oxidation of -glutamyl- reported for ovothiols. Currently, industrial-scale production cysteine (γ-Glu-Cys) to γ-glutamyl-cysteine sulfinic acid.27 for both ergothioneine and ovothiol has been proven to be − Built upon this M. thermoresistibile EgtB crystal structure, three challenging.16,40 42 Therefore, biosynthesis was explored as an EgtB computational mechanistic studies were reported alternative option. In recent years, synergistic work between recently, using density function theory or quantum mechan- the Seebeck and Liu laboratories has led to the full elucidation 28,29,44 of the aerobic ergothioneine and ovothiol biosynthetic ics/molecular mechanics (QM/MM) methods. Con- 12−29 clusions from these reports differ in at least two aspects. First, pathways. In these two pathways, a sulfur atom is fi transferred from L-Cys to an L-His side-chain by a combination there are two possibilities for the rst half of this four-electron of two reactions: an oxidative C−S bond formation (OvoA in oxidation process: the sulfur oxidation suggested by Tian, Wei, ovothiol, Egt1/EgtB in ergothioneine biosynthesis, Scheme 1) and a reductive C−S lyase reaction (OvoB in ovothiol, and Received: September 27, 2018 EgtE/Egt2 in ergothioneine biosynthesis, Scheme 1). In Revised: November 12, 2018 ergothioneine and ovothiol biosynthesis, the sulfoxide Published: December 7, 2018

Scheme 1. Ergothioneine and Ovothiol A Biosyntheses: (A) Owing to the lack of an OvoA crystal structure, we created Ovothiol A Biosynthetic Pathway; (B) Two Aerobic an OvoA homology model (Figure S1), which suggested that Ergothioneine Biosynthetic Pathways; and (C) Two OvoA Y417 is the counterpart of EgtB Y377 (Figure 1B). Potential Pathways for the Oxidative C−S Bond Formation Indeed, in our recent report,14 by replacing Y417 in OvoA with in OvoA-Catalysis 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 (eq 1, Figure 1D). However, because tyrosine and ff 45−48 MtTyr 14 di er in both pKa and reduction potentials, 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 analogue, 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 (eq 1, Figure 1D) enables the use of kinetic isotope effect (KIE) studies via two different approaches: kinetic isotope sensitive branching (eq 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 four- electron oxidation process. The Methanococcus jannaschii tyrosyl amber suppressor Tyr tRNA (MjtRNA CUA)/tyrosyl-tRNA synthetase (MjTyrRS) pair has been developed to specifically recognize MeOTyr.45 MeOTyr was synthesized (Figures S2 and S3)and incorporated into OvoA to produce OvoAY417MeOTyr (MeOTyr-OvoA).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 equiv of iron using atomic emission spectroscopy (Figure S4).56,57 The MeOTyr- OvoA was then characterized by oxygen consumption, 1H NMR, and 13C NMR 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 Figure 1. Key information related to the experimental design of the −1 ± μ ± μ 0.1 s and KM of 399 36 M for L-His, KM of 175 19 M present study. (A) Reported EgtB·γGC·hercynine complex (PDB: for L-Cys (Figure S6). The KMs of both L-His and L-Cys of the 4X8D). (B) Predicted OvoA structure (Figure S1 for more detail). MeOTyr OvoA variant are close to that of wild-type OvoA (C) Tyrosine-cysteine cross-linking in cysteine dioxygenase and two ± μ ± μ (KM of 420 31 M for L-His, and KM of 300 34 M for L- tyrosine analogues (MtTyr and MeOTyr). (D) Schematic represen- 19 ∼ tation of isotope sensitive branching method and equations for Cys) reported previously, while kcat is 6.6-fold lower than ± −1 1 calculating kinetic isotope effects (KIEs). that of wild type OvoA (kcat of 9.53 0.33 s ). In the H 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 and co-workers (pathway I, Scheme 1C)29,44 or the oxidative NMR spectra indicated that cysteine sulfinic acid 13 comprises C−S bond formation proposed by Faponle et al. (pathway II, up to ∼26% of the product mixture (Figures 2, S8 and S9). In Scheme 1C).28 Second, the EgtB Y377 could either function as wild type OvoA, cysteine sulfinic acid 13 is observed as less a Lewis acid/base as proposed by Tian et al.,29,44 or it could be than 10% of the product mixture.17 These results indicate that a part of a proton-coupled electron transfer (PCET) process as substitution of Y417 by MeOTyr in OvoA does not affect the suggested by Faponle et al.28 In the analogous OvoA-catalytic identities of the products from OvoA catalysis; rather, this process, based on these two EgtB-mechanistic models, OvoA mutation alters the partitioning between oxidative coupling reaction may proceed via either pathway I (intermediate 10)or and cysteine sulfinic acid formation pathways (eq 1 of Figure pathway II (intermediate 11 or 12, Scheme 1C). 1D). This result differs from the case of using hercynine as the

254 DOI: 10.1021/acscatal.8b03903 ACS Catal. 2019, 9, 253−258 ACS Catalysis Letter

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 −2 unlabeled L-His and [U H5]-His as the substrate (Figure 3A). In this method, three parameters need to be measured

Figure 2. Measuring kinetic isotope sensitive branching KIE using 2 [U- H5]-His as the substrate. (A) Conditions used for the two reactions. Mass spectrometry spectra of (B) coupling products 2/2b and (C) sulfinic acids 13/13b. The ratios of compounds in the two sets of reactions (B,C) were used to calculate the KIE for k3 step at the branching point. substrate to replace histidine, where the C−S bond formation regioselectivity changes from the histidine δ-position to the ε- position.19 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 L- 2 β 13 Cys were used as substrates, while [U- H5]-His and [ - C]- Figure 3. Substrate V/K KIE measurement in an intermolecular Cys were the substrates for the second set of reaction (Figure competition experiment (A) Schematic representation of the 2A). These two reactions were quenched and mixed, and the experimental design and equations used for data analysis. L-His 1 −2 ratios between the two products (2/2b and 13/13b) were and [U H5]- His 1b were mixed at a given ratio and L-Cys was then fi introduced to initiate the reaction. After the reaction was quenched at quanti ed by mass spectrometry; the analytical results from ff − −15 one set of experiments are presented in Figure 2. The ratio of 2 di erent time points (e.g., f = 0.6 0.8), [U N3]-His 1c of known concentration was then added to the reaction and the amount of and 2b ([P1]H/[P1]D, Figure 2B) is 0.68. In the same remaining 1 and 1b was calculated on the basis of their ion intensities experiment, the ratio between 13 and 13b ([P2]H/ [P2]D, relative to that of 1c. Using the same approach, the ratio between 1 Figure 2C) is 0.81. Based on the kinetic model shown in and 1b prior to initiation of the reaction was also measured (f = 0). −2 Figure 1D, the KIE obtained for [U H5]-His is 0.84 (eq 2, For the equation (eq 9 in part A) used to calculate deuterium KIE on Figure 1D). This experiment was repeated at least four times, V/K, the meanings of these terms are f (percent conversion), X1 and the average KIE was 0.86 ± 0.03. This inverse KIE for (initial mole fraction of the unlabeled substrate 1), X1b (initial mole 2 ff [U- H5]-His observed in MeOTyr-OvoA (Figure 2)diers fraction of the labeled substrate 1b), R0 (initial substrate ratio significantly from that of wild type OvoA, in which the KIE for between 1b and 1), Rs (residual substrate ratio between 1b and 1 at 2 14 ff time point t). (B) Representative mass spectrometry results for one [U- H5]-His is close to unity. Such di erences imply that substitution of Y417 by MeOTyr changes the rate-determining set of experiment at f = 0. (C) Representative mass spectrometry 2 results for the same set of experiment at f = 0.704. step in OvoA-catalysis. In this experiment, [U- H5]-His was used as the substrate, and we will discuss this in later sections. The observed KIE is most likely a secondary deuterium KIE. It is unlikely that α and β-positions will have significant (eq 9, Figure 3): the percent conversion (f) of the reaction at contribution to this KIE. However, the measured KIE may the time point t and the ratio between the heavy and light ff ε δ be a combined e ect from both - and -positions of the forms of the substrate of interest (Rs and R0 are the ratios of δ −2 imidazole side-chain, whereas the -position might be the [U H5]-His 1b and unlabeled L-His 1 at time point t and at major contributor to the observed inverse secondary deuterium time point zero, respectively, Figure 3A). To accurately KIE. measure the percent conversion f and the ratio between 1b In oxygenases and oxidases, in the vast majority of cases, O2 and 1 at the zero point and time t, another form of L-His, fi 15 activation and the formation of oxidative species is the rst [U- N3]-His 1c was introduced. A known concentration of 1c irreversible step. Due to these reasons, it is not possible to was mixed with the reaction mixture at the zero point in a measure the isotope effect on V/K. However, the presence of certain ratio and the mixture was analyzed by mass branching pathway unmasks the isotope effect on V/K (DV/K, spectrometry. With the known concentration of 1c, and the − eq 3, Figure 1D), which makes the measurement feasible.50 55 measured ion intensities of 1, 1b, and 1c, the initial ratio of 1 Therefore, in OvoA-studies, besides the use of isotopically and 1b can be calculated (Figure 3A). Similarly, at time point t, sensitive branching method to directly measure the isotope an aliquot of the reaction mixture was withdrawn and effect on one of the steps at the branching point, we also quenched by hydrochloric acid. The quenched reaction measured deuterium isotope effect on V/K to provide another mixture was then mixed with a known amount of 1c and

255 DOI: 10.1021/acscatal.8b03903 ACS Catal. 2019, 9, 253−258 ACS Catalysis Letter characterized by mass spectrometry to determine the ratio IB, Fe(IV)O oxidizes the imidazole side-chain to produce a between 1 and 1b at time point t (Figure 3A). cation radical 18, which then combines with sulfenic acid to With the accurately measured R0, Rs, and percent conversion form the C−S bond in 19. In this mechanism, the active site f, all of the needed parameters related to this competition tyrosine functions as a base to deprotonate 19 to produce 2, 53 experiment can be obtained (eqs 4−8, Figure 3A), which and this is the rate-limiting step in EgtB-catalysis based on the D were then used to calculate deuterium KIE V/K (eq 9, Figure computational studies reported by Wei et al.29 In pathway II, 3A). The reaction was quenched at time points when the Faponle et al. suggested that the active site tyrosine is part of a percent conversion f is in the range of 0.6−0.8 to minimize 54,55 proton-coupled electron transfer process and in this mecha- errors associated with KIE measurement. Panels B and C nism, oxidative C−S bond formation to form intermediate 21 of Figure 3 are representative spectra at the zero time point is the first half-reaction of the overall four-electron oxidation and at the time point t (f = 70.4%) for one set of experiment. 28 process. In addition, Faponle et al. suggested that the 21 → Detailed calculations for this set of experiments are shown in 22 reaction is the rate-limiting step and inverse deuterium Figure S10. The experiment was repeated at least four times, ff δ D ± isotopic e ect is predicated if the -hydrogen is replaced with and the V/K obtained was 0.94 0.02, which is also an ff inverse deuterium KIE. deuterium (secondary deuterium isotope e ect). If the kinetic model shown in eq 1 of Figure 1D does Based on the above discussion of the two mechanistic represent OvoA-catalysis, the KIE measured from kinetic models, in pathway I, the active site tyrosine residue most isotope sensitive branching (Figure 2 studies) should be likely functions as a Lewis base and primary deuterium isotope ff δ 2 correlated with KIE in the competition experiment (Figure 3 e ect as high as 5.7 for [ - H]-His has been predicted by Wei 29 studies) using eq 3 of Figure 1D. In eq 3, k3H/k5 is the ratio et al. In pathway II, however, Tyr417 is involved in redox (74:26) of compound 2 to 13 in the reaction using unlabeled chemistry and an inverse secondary deuterium isotope effect is D δ 2 28 L-His and L-Cys. By incorporating k3 measured in Figure 2 predicted for [ - H]-His by Faponle et al. In this study, when studies into eq 3, the calculated deuterium KIE on V/K [D(V/ a MeOTyr-OvoA variant is used as the catalyst, indeed, an K)] is 0.96 ± 0.01 (Figure S10). The experimentally measured inverse deuterium KIE was observed from studies using both D(V/K) from Figure 3 studies is 0.94 ± 0.02. There is a kinetic isotope sensitive branching and intermolecular reasonably good agreement between these two sets of competition methods. This result differs from that of wild independent studies. type OvoA,14 which has a KIE close to unity. Therefore, the When Seebeck and co-workers first reported their discovery replacement of Y417 with MeOTyr has led to a change of the of the OvoA enzyme, they proposed several possible catalytic rate-limiting step in OvoA-catalysis. MeOTyr-OvoA produces 21 mechanism. Three computational EgtB mechanistic studies the same two products as that of wild type OvoA, which highly 28,29,44 were reported recently. Because of the similarity suggests that mechanistically, MeOTyr-OvoA behaves the between EgtB- and OvoA-catalysis, we included three OvoA- same as wild type OvoA. However, the ratio of the two mechanistic models here based on the mechanistic models products (2 and 13, Scheme 2) changes when Y417 is replaced proposed from EgtB-computational work (Scheme 2). For by an unnatural tyrosine. MeOTyr has a pKa that is almost the same as that of Tyr, while their reduction potentials are Scheme 2. Proposed OvoA Mechanistic Models 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 evidence (an inverse ff −2 deuterium isotope e ect for [U H5]-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 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. fi pathways IA and IB, the rst half of the reaction is the ■ ASSOCIATED CONTENT oxidation of cysteine to a sulfenic acid intermediate (10, * Scheme 2). Based on the computational studies from Tian et S Supporting Information al., Fe(IV)O species 10 may deprotonate imidazole δ- The Supporting Information is available free of charge on the position to generate an anion 17, which nucleophilically ACS Publications website at DOI: 10.1021/acscatal.8b03903. attacks the sulfenic acid to produce sulfoxide 2. In this mechanistic model, Tian et al. also suggested an alternative Experimental details, protein characterizations, NMR pathway using the active site tyrosine as the base for a and MS spectra of reactions, and kinetic study results deprotonation process (species 19, Scheme 2).44 In pathway (PDF)

256 DOI: 10.1021/acscatal.8b03903 ACS Catal. 2019, 9, 253−258 ACS Catalysis Letter ■ AUTHOR INFORMATION (14) Chen, L.; Naowarojna, L.; Song, H.; Wang, S.; Wang, J.; Deng, Corresponding Authors Z.; Zhao, C.; Liu, P. Use of a Tyrosine Analog to Modulate the Two Activities of a Non-heme Iron Enzyme OvoA in Ovothiol Biosyn- *E-mail: [email protected]. * thesis, Cysteine Oxidation Versus Oxidative C-S Bond Formation. J. E-mail: [email protected]. Am. Chem. Soc. 2018, 140 (13), 4604−4612. ORCID (15) Song, H.; Hu, W.; Naowarojna, N.; Her, A. S.; Wang, S.; Desai, Pinghua Liu: 0000-0002-9768-559X R.; Qin, L.; Chen, X.; Liu, P. Mechanistic Studies of a Novel C-S Author Contributions Lyase in Ergothioneine Biosynthesis: the Involvement of a Sulfenic ⊥ (L.C., N.N.) These authors contributed equally. Acid Intermediate. Sci. Rep. 2015, 5, 11870. (16) Xu, J. Z.; Yadan, J. C. Synthesis of L-(+)-Ergothioneine. J. Org. Notes Chem. 1995, 60 (20), 6296−6301. The authors declare no competing ﬁnancial interest. (17) Song, H.; Her, A. S.; Raso, F.; Zhen, Z.; Huo, Y.; Liu, P. Cysteine Oxidation Reactions Catalyzed by a Mononuclear Non- ■ ACKNOWLEDGMENTS heme Iron Enzyme (OvoA) in Ovothiol Biosynthesis. Org. Lett. 2014, − This work is supported in part by grant from the National 16 (8), 2122 5. Science Foundation (CHE-1309148 to P.L.), grants from (18) Hu, W.; Song, H.; Sae Her, A.; Bak, D. W.; Naowarojna, N.; National Natural Science Foundation of China (31670030 to Elliott, S. J.; Qin, L.; Chen, X.; Liu, P. Bioinformatic and Biochemical Characterizations of C-S Bond Formation and Cleavage Enzymes in C.Z., 31628004 to J.W.) and grant from China Postdoctoral the Fungus Neurospora Crassa Ergothioneine Biosynthetic Pathway. Science Foundation (2018M642901 to L.C.). C.Z. is Org. Lett. 2014, 16 (20), 5382−5. supported by Young Talents Program of Wuhan University. (19) Song, H.; Leninger, M.; Lee, N.; Liu, P. 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258 DOI: 10.1021/acscatal.8b03903 ACS Catal. 2019, 9, 253−258