Kinetic Mechanism of the Dechlorinating Flavin-dependent Monooxygenase HadA.
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 organismRalstonia pickettiiDTP0602, 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 that HadA prefers 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.
From the Department of Biochemistry and Center for Excellence in Protein and Enzyme Technology, Faculty of Science, Mahidol University, Bangkok 10400 and.
Panu
P
Pimviriyakul
the Department of Biochemistry, Faculty of Dentistry, Chulalongkorn University, Bangkok 10300, Thailand.
Kittisak
K
Thotsaporn
the Department of Biochemistry, Faculty of Dentistry, Chulalongkorn University, Bangkok 10300, Thailand.
Jeerus
J
Sucharitakul
From the Department of Biochemistry and Center for Excellence in Protein and Enzyme Technology, Faculty of Science, Mahidol University, Bangkok 10400 and pimchai.cha@mahidol.ac.th.
Pimchai
P
Chaiyen
D002733:Chlorophenols / Q000737:chemistry / Q000378:metabolism*; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism*; D016905:Gram-Negative Bacterial Infections / Q000382:microbiology; D054879:Halogenation; D006801:Humans; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000378:metabolism*; D049920:Ralstonia pickettii / Q000737:chemistry / Q000201:enzymology* / Q000378:metabolism; D013379:Substrate Specificity
D002733:Chlorophenols; D005182:Flavin-Adenine Dinucleotide; D006899:Mixed Function Oxygenases; C099366:chlorophenol 4-monooxygenase
chlorophenol; enzyme kinetics; flavin; flavin adenine dinucleotide (FAD); hydroxylase; monooxygenase; pre-steady-state kinetics