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Hexachlorobenzene catabolism involves a nucleophilic aromatic substitution and flavin-N5-oxide formation Sanjoy Adak, and Tadhg P. Begley Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.9b00012 • Publication Date (Web): 31 Jan 2019 Downloaded from http://pubs.acs.org on February 3, 2019
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Biochemistry
Hexachlorobenzene catabolism involves a nucleophilic aromatic substitution and flavin-N5-oxide formation Sanjoy Adak and Tadhg P. Begley* Department of chemistry, Texas A&M University, 3255 TAMU, College station, TX 77843, USA. Supporting Information Placeholder ABSTRACT: HcbA1 is a unique flavoenzyme that catalyzes the first step in the bacterial hexachlorobenzene catabolic pathway. Here we report in vitro reconstitution of the HcbA1catalyzed reaction. Detailed mechanistic studies provide evidence for nucleophilic aromatic substitution and flavin-N5oxide formation.
Hexachlorobenzene (HCB, 5) was widely used as a fungicide in agriculture until it was banned in 2001 under the Stockholm convention on persistent organic pollutants.1 During the development of a bioremediation strategy for this compound, an HCB catabolizing strain of Nocardioides (strain PD653) was isolated and the catabolic operon involved in the conversion of HCB to pentachlorophenol (PCP, 9) was cloned and sequenced.2, 3 This gene cluster encoded three genes - a flavin monooxygenase (HcbA1), and two flavin reductases (HcbA2 and HcbA3). Cell lysate containing HcbA1 and HcbA3 catalyzed the conversion of HCB to PCP. We have previously characterized the mechanism of dibenzothiophene sulfone mono-oxygenase (DszA).4 For this enzyme, flavin hydroperoxide (1) adds to a substrate electro
philic carbon. This is followed by flavin elimination to give the substrate hydroperoxide which then reacts with the eliminated flavin to give a flavin-N5-oxide (4) and the product. This mechanism represents a general reaction motif that can be used to predict flavin-N5-oxide utilizing enzymes (Figure 1A). Guided by this hypothesis, we have recently reported a second example of a flavin-N5-oxide-utilizing enzyme – uracil monooxygenase (RutA)5, 6 involved in an oxidative amide cleavage in uracil catabolism. We, therefore, considered the mechanism shown in Figure 1B for hexachlorobenzene mono-oxygenase (HcbA1) as a possibility. In this proposal, flavin hydroperoxide adds to the highly electron depleted benzene ring to give the Meisenheimer intermediate 6. Loss of chloride followed by flavin elimination gives the peroxide 8. Cleavage of this peroxide by the eliminated flavin gives the final product 9 and flavin-N5-oxide. In this paper, a mechanistic study that supports this proposal for the HcbA1-catalyzed reaction is described. The HcbA1 sequence was optimized for expression in E.coli and inserted into the pTHT expression vector (derivative of pET28b vector with a TEV protease cleavage site after the Nterminal His-tag). The resulting plasmid was transformed into E.coli BL21(DE3). Overexpressed protein was purified using
Figure 1: A) A general model for the prediction of enzymes that generate flavin-N5-oxide as an intermediate. B) Mechanistic proposal for the conversion of HCB to PCP.
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Figure 2: Reconstitution of the HcbA1-catalyzed reaction. A) HPLC chromatogram of reaction mixture showing formation of a new peak eluting at 3.0 min. B) MS data confirming PCP as the reaction product. C) Coelution of the HcbA1 reaction product with an authentic PCP standard. D) The reaction catalyzed by HcbA1. E) MS analysis of the PCP formed when the reaction was run in H218O. F) MS analysis of the PCP formed when the reaction was run in 18O2. Ni-NTA-affinity chromatography. The HcbA1-catalyzed reaction was reconstituted by incubating HCB, HcbA1, E.coli flavin reductase (Fre)5, FMN (not FAD), and NADH at 25o C for 3hr. Under these conditions, HCB was converted to a new product (Figure 2A) which was identified as PCP by MS analysis and by HPLC comigration with an authentic sample (Figure 2B and 2C). The mechanistic proposal shown in Figure 1B predicts that the oxygen atom in PCP is derived from molecular oxygen. This was confirmed by MS analysis of the product formed when the reaction was run in the presence of 18O2 (Figure 2F). In a control reaction run in H218O, 18O incorporation into PCP was not detected (Figure 2E).
Figure 3: Characterization of flavin-N5-oxide in the HcbA1catalyzed reaction. A) EIC at m/z = 473.1 corresponding to [M+H]+ for FMN-N5-oxide. B) Exact mass of the FMN-N5-oxide formed when the reaction was carried out in the presence of 16O . C) Exact mass of the FMN-N5-oxide formed when the 2 reaction was carried out in the presence of 18O2.
A similar strategy to that developed for the DszA and RutA systems was used to test for flavin-N5-oxide formation in the HcbA1-catalyzed reaction. To avoid reduction of the flavin-N5oxide, the HcbA1-catalyzed reaction was carried out using photo-reduced FMN. A typical reaction mixture containing stoichiometric amounts of HCB, HcbA1 and FMNH2 was incubated for 30 min under anaerobic conditions before exposed to atmospheric oxygen for 1hr to allow the formation of the flavin-C4a-peroxide and the subsequent oxidation reaction to take place. LC-MS analysis of the resulting reaction mixture demonstrated the formation of a product with a mass corresponding to flavin-N5-oxide (Figure 3A and 3B). When the reaction was carried out in the presence of 18O2, the predicted 2 Da increase in the mass of flavin-N5-oxide was observed (Figure 3C). Enzyme-mediated aryl ring dehalogenation reactions have been the focus of considerable study over the past several decades.7-9 Two systems, relevant to the HCB dehalogenation, are shown in Figure 4. In the 4-chlorobenzoyl-CoA dehalogenase-catalyzed reaction10-14 (panel A), an active site carboxylate adds to C4 of the substrate to give the Meisenheimer complex 11. Loss of chloride followed by ester hydrolysis completes the reaction. Flavin hydroperoxide functions as an electrophile mediating the hydroxylation of the para position of 9 in the pentachlorophenol dehalogenasecatalyzed reaction15-17 (panel B). Loss of chloride completes the reaction. The HCB dehalogenase-catalyzed reaction combines features of these two reactions in a novel way using flavin hydroperoxide to form a Meisenheimer complex and forming flavin-N5-oxide as a new intermediate in enzymatic dehalogenation reactions. The identification of HcbA1 as a flavin-N5-oxide-utilizing enzyme further validates the proposal that flavin-N5-oxides are likely intermediates in flavoenzymes that form substrate hydroperoxides in the absence of an active site cysteine (Figure 1A).18 This model is consistent with three of the four known flavin-N5-oxide-utilizing enzymes. It does not include EncM1925 and the full scope of nature’s use of this new flavin oxidation state has not yet been uncovered.
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Biochemistry
Figure 4: Relevant halobenzene dehalogenation reactions. A) The 4-chlorobenzoyl-CoA dehalogenase-catalyzed reaction that proceeds via a Meisenheimer intermediate. B) The PcpBcatalyzed conversion of pentachlorophenol to tetrachlorobenzoquinone.
ASSOCIATED CONTENT Supporting Information Detailed procedure for overexpression and purification of HcbA1, HPLC and LC-MS parameters, enzymatic reaction conditions AUTHOR INFORMATION
Corresponding Author *
[email protected] Funding Sources This research was supported by the Robert A. Welch Foundation Grant-A0034.
Notes The authors declare no competing financial interests.
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[6] Adak, S., and Begley, T. P. (2017) Flavin-N5-oxide: A new, catalytic motif in flavoenzymology, Arch Biochem Biophys 632, 4-10. [7] Fetzner, S. (1998) Bacterial dehalogenation, Appl. Microbiol. Biotechnol. 50, 633-657. [8] de Jong, R. M., and Dijkstra, B. W. (2003) Structure and mechanism of bacterial dehalogenases: different ways to cleave a carbon-halogen bond, Curr. Opin. Struct. Biol. 13, 722-730. [9] Agarwal, V., Miles, Z. D., Winter, J. M., Eustaquio, A. S., El Gamal, A. A., and Moore, B. S. (2017) Enzymatic Halogenation and Dehalogenation Reactions: Pervasive and Mechanistically Diverse, Chem. Rev. (Washington, DC, U. S.) 117, 5619-5674. [10] Benning, M. M., Taylor, K. L., Liu, R.-Q., Yang, G., Xiang, H., Wesenberg, G., Dunaway-Mariano, D., and Holden, H. M. (1996) Structure of 4-chlorobenzoyl coenzyme A dehalogenase determined to 1.8 Å resolution: An enzyme catalyst generated via adaptive mutation, Biochemistry 35, 8103-8109. [11] Luo, L., Taylor, K. L., Xiang, H., Wei, Y., Zhang, W., and DunawayMariano, D. (2001) Role of Active Site Binding Interactions in 4Chlorobenzoyl-Coenzyme A Dehalogenase Catalysis, Biochemistry 40, 15684-15692. [12] Dong, J., Carey, P. R., Wei, Y., Luo, L., Lu, X., Liu, R.-Q., and DunawayMariano, D. (2002) Raman Evidence for Meisenheimer Complex Formation in the Hydrolysis Reactions of 4-Fluorobenzoyl- and 4Nitrobenzoyl-Coenzyme A Catalyzed by 4-Chlorobenzoyl-Coenzyme A Dehalogenase, Biochemistry 41, 7453-7463. [13] Zhang, W., Wei, Y., Luo, L., Taylor, K. L., Yang, G., Dunaway-Mariano, D., Benning, M. M., and Holden, H. M. (2001) Histidine 90 Function in 4Chlorobenzoyl-Coenzyme A Dehalogenase Catalysis, Biochemistry 40, 13474-13482. [14] Lau, E. Y., and Bruice, T. C. (2001) The active site dynamics of 4chlorobenzoyl-CoA dehalogenase, Proc. Natl. Acad. Sci. U. S. A. 98, 95279532. [15] Crawford, R. L., Jung, C. M., and Strap, J. L. (2007) The recent evolution of pentachlorophenol (PCP)-4-monooxygenase (PcpB) and associated pathways for bacterial degradation of PCP, Biodegradation 18, 525-539. [16] Hlouchova, K., Rudolph, J., Pietari, J. M. H., Behlen, L. S., and Copley, S. D. (2012) Pentachlorophenol Hydroxylase, a Poorly Functioning Enzyme Required for Degradation of Pentachlorophenol by Sphingobium chlorophenolicum, Biochemistry 51, 3848-3860. [17] Yadid, I., Rudolph, J., Hlouchova, K., and Copley, S. D. (2013) Sequestration of a highly reactive intermediate in an evolving pathway for degradation of pentachlorophenol, Proc Natl Acad Sci U S A 110, E2182-2190. [18] Hall, A., Karplus, P. A., and Poole, L. B. (2009) Typical 2-Cys peroxiredoxins - structures, mechanisms and functions, FEBS J. 276, 2469-2477. [19] Teufel, R., Miyanaga, A., Michaudel, Q., Stull, F., Louie, G., Noel, J. P., Baran, P. S., Palfey, B., and Moore, B. S. (2013) Flavin-mediated dual oxidation controls an enzymatic Favorskii-type rearrangement, Nature 503, 552-556. [20] Teufel, R., Stull, F., Meehan, M. J., Michaudel, Q., Dorrestein, P. C., Palfey, B., and Moore, B. S. (2015) Biochemical Establishment and Characterization of EncM's Flavin-N5-oxide Cofactor, J Am Chem Soc 137, 8078-8085. [21] Teufel, R., Agarwal, V., and Moore, B. S. (2016) Unusual flavoenzyme catalysis in marine bacteria, Curr Opin Chem Biol 31, 3139. [22] Teufel, R. (2017) Flavin-catalyzed redox tailoring reactions in natural product biosynthesis, Arch Biochem Biophys 632, 20-27. [23] Teufel, R. (2018) Preparation and Characterization of the Favorskiiase Flavoprotein EncM and Its Distinctive Flavin-N5-Oxide Cofactor, Methods Enzymol 604, 523-540. [24] Saleem-Batcha, R., Stull, F., Sanders, J. N., Moore, B. S., Palfey, B. A., Houk, K. N., and Teufel, R. (2018) Enzymatic control of dioxygen binding and functionalization of the flavin cofactor, Proc Natl Acad Sci U S A 115, 4909-4914. [25] Saleem-Batcha, R., and Teufel, R. (2018) Insights into the enzymatic formation, chemical features, and biological role of the flavin-N5-oxide, Curr. Opin. Chem. Biol. 47, 47-53.
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UniProt accession ID of HcbA1: A0A1V1W347
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