A Monooxygenase Catalyzes Sequential Dechlorinations of 2,4,6-Trichlorophenol by Oxidative and Hydrolytic Reactions

Abstract

Ralstonia eutropha JMP134 2,4,6-trichlorophenol (2,4,6-TCP) 4-monooxygenase catalyzes sequential dechlorinations of 2,4,6-TCP to 6-chlorohydroxyquinol. Although 2,6-dichlorohydroxyquinol is a logical metabolic intermediate, the enzyme hardly uses it as a substrate, implying it may not be a true intermediate. Evidence is provided to support the proposition that the monooxygenase oxidized 2,4,6-TCP to 2,6-dichloroquinone that remained with the enzyme and got hydrolyzed to 2-chlorohydroxyquinone, which was chemically reduced by ascorbate and NADH to 6-chlorohydroxyquinol. When the monooxygenase oxidized 2,6-dichlorophenol, the product was 2,6-dichloroquinol, which was not further converted to 6-chlorohydroxyquinol, implying that the enzyme only converts 2,6-dichloroquinone to 6-chlorohydroxyquinol. Stoichiometric analysis indicated the consumption of one O 2 molecule per 2,4,6-TCP converted to 6-chlorohydroxyquinol, ruling out the possibility of two oxidative reactions. Experiments with 18 Olabeling gave direct evidence for the incorporation of oxygen from both O 2 and H 2 O into the produced 6-chlorohydroxyquinol. A monooxygenase that catalyzes hydroxylation by both oxidative and hydrolytic reactions has not been reported to date. The ability of the enzyme to perform two types of reactions is not due to the presence of a second functional domain but rather is due to catalytic promiscuity, as a homologous monooxygenase converts 2,4,6-TCP to only 2,6-dichloroquinol. Employing both conventional catalysis and catalytic promiscuity of a single enzyme in two consecutive steps of a metabolic pathway has been unknown previously.

Kinetic Mechanism of the Dechlorinating Flavin-dependent Monooxygenase HadA

“…HadA has high sequence identity to TcpA from R. eutropha JMP134 (86.7%). Previously, analysis of TcpA substrate utilization was mainly done with 2,4,6-TCP, and 2,4,6-TCP was specified as a native substrate for TcpA (11)(12)(13). Previous studies of TftD from B. cepacia AC1100 (64.2% identity to HadA) have compared substrate preference between 2,4,5-TCP and 2,4,6-TCP.…”

“…However, turnover of PcpB using PCP as a substrate is very slow, implying that PCP may not be a native substrate of PcpB (18). Many oxidative dechlorination reactions are also catalyzed by two-component flavindependent enzymes, including TftD from B. cepacia AC1100 (9,10), TcpA from R. eutropha JMP134 (13), and HadA from R. pickettii DTP0602 (15). Although a few of these enzymes have been shown to catalyze oxidative dechlorination of CPs, their reaction mechanisms are not well understood.…”

“…None of these enzymes have been explored with regard to their kinetic mechanisms, and the rate constants associated with the individual steps have yet to be measured. Only X-ray structures of TftD and TcpA and the overall bioconversion activities of these enzymes have been reported (9,10,13,15,21).…”

“…Degradation of CPs such as 2,4,5-TCP, 2,4,6-TCP, or PCP can be carried out by various enzymatic systems such as tft clusters from Burkholderia cepacia AC1100 (9,10), tcp clusters from Ralstonia eutropha JMP134 (11)(12)(13), had clusters from Ralstonia pickettii DTP0602 (14 -16), and pcp clusters from Sphingobium chlorophenolicum (17)(18)(19).…”

A Novel Gene, Encoding 6-Hydroxy-3-Succinoylpyridine Hydroxylase, Involved in Nicotine Degradation by Pseudomonas putida Strain S16

“…Based on the analysis of pathway, the process began with an enzymecatalyzed dehydrogenation, followed by spontaneous hydrolysis and then a repeat of the enzyme-catalyzed dehydrogenation to produce SP. Similar cases were found in Arthrobacter for L-6-hydroxynicotine oxidase or D-6-hydroxynicotine oxidase in the nicotine degradation pathway (5) and a monooxygenase catalyzing sequential dechlorinations of 2,4,6-trichlorophenol by oxidative and hydrolytic reactions (42). SP might then be hydroxylated at position 6 of the pyridine ring to generate HSP by a monooxygenase, and HSP was transformed to DHP by HSP hydroxylase.…”