Publication: Kinetic mechanism of the dechlorinating flavin-dependent monooxygenase HadA
Issued Date
2017-03-24
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ISSN
1083351X
00219258
00219258
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2-s2.0-85016273515
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Mahidol University
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SCOPUS
Bibliographic Citation
Journal of Biological Chemistry. Vol.292, No.12 (2017), 4818-4832
Suggested Citation
Panu Pimviriyakul, Kittisak Thotsaporn, Jeerus Sucharitakul, Pimchai Chaiyen Kinetic mechanism of the dechlorinating flavin-dependent monooxygenase HadA. Journal of Biological Chemistry. Vol.292, No.12 (2017), 4818-4832. doi:10.1074/jbc.M116.774448 Retrieved from: https://repository.li.mahidol.ac.th/handle/123456789/41945
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Title
Kinetic mechanism of the dechlorinating flavin-dependent monooxygenase HadA
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Abstract
© 2017 by The American Society for Biochemistry and Molecular Biology, Inc. The accumulation of chlorophenols (CPs) in the environment, due to their wide use as agrochemicals, has become a serious environmental problem. These organic halides can be degraded by aerobic microorganisms, where the initial steps of various biodegradation pathways include an oxidative dechlorinating process in which chloride is replaced by a hydroxyl substituent. Harnessing these dechlorinating processes could provide an opportunity for environmental remediation, but detailed catalytic mechanisms for these enzymes are not yet known. To close this gap, we now report transient kinetics and product analysis of the dechlorinating flavin-dependent monooxygenase, HadA, from the aerobic organism Ralstonia pickettii DTP0602, identifying several mechanistic properties that differ from other enzymes in the same class. We first overexpressed and purified HadA to homogeneity. Analyses of the products from single and multiple turnover reactions demonstrated thatHadAprefers 4-CP and 2-CP over CPs with multiple substituents. Stopped-flow and rapid-quench flow experiments of HadA with 4-CP show the involvement of specific intermediates (C4a-hydroperoxy-FAD and C4a-hydroxy-FAD) in the reaction, define rate constants and the order of substrate binding, and demonstrate that the hydroxylation step occurs prior to chloride elimination. The data also identify the non-productive and productive paths of the HadA reactions and demonstrate that product formation is the rate-limiting step. This is the first elucidation of the kinetic mechanism of a two-component flavin-dependent monooxygenase that can catalyze oxidative dechlorination of various CPs, and as such it will serve as the basis for future investigation of enzyme variants that will be useful for applications in detoxifying chemicals hazardous to human health.