Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: KEGG:D02011 (FAD)
 5,530 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Burkholderia cepacia strain AC1100 can be induced for the degradation of 2,4,5-trichlorophenol (2,4,5-TCP). We have purified the active enzyme 30-fold to apparent homogeneity with a 44% yield by a two-step chromatographic procedure, and showed that it consists of a single type of subunit of 59 kDa based on SDS-PAGE using Coomassie blue and Sypro staining. This enzyme has no bound prosthetic group but requires exogenous addition of FAD and NADH to perform the dioxygen-dependent hydroxylation in the 4-position of 2,4,6-TCP. Studies of the stoichiometry revealed the consumption of 2 mol of NADH plus 1 mol of dioxygen per mol of 2,4,6-TCP with identification of the reaction product as 2,6-dichlorohydroquinone. Steady state kinetic parameters for cofactors and a variety of substrates were determined. Low K(m) values of 1+/-0.1 microM, 32+/-5 microM and 4+/-2 microM were found for FAD, NADH and 2,6-dichlorophenol (2,6-DCP), respectively, under saturating conditions for the two others. In the presence of 2,6-DCP as a substrate, methimazole (MMI) inhibited the enzyme competitively with a K(i)=27 microM. When other polychlorinated substrates were studied, IC(50) values for MMI were found in a range compatible with their apparent affinity. On the basis of aromatic product formation, NADH and O(2) consumption schemes for 2,4,6-TCP and 2,4,5-TCP degradation are discussed. A Blast search revealed that this enzyme has a high sequence identity (60%) with 2,4,6-TCP-4-monooxygenases from Burkholderia pickettii and from Azotobacter sp. strain GP1 which all of them catalyze para hydroxylative dehalogenation.
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PMID:Purification and catalytic properties of the chlorophenol 4-monooxygenase from Burkholderia cepacia strain AC1100. 1141 Feb 85

Ralstonia eutropha JMP134 can grow on several chlorinated aromatic pollutants, including 2,4-dichlorophenoxyacetate and 2,4,6-trichlorophenol (2,4,6-TCP). Although a 2,4,6-TCP degradation pathway in JMP134 has been proposed, the enzymes and genes responsible for 2,4,6-TCP degradation have not been characterized. In this study, we found that 2,4,6-TCP degradation by JMP134 was inducible by 2,4,6-TCP and subject to catabolic repression by glutamate. We detected 2,4,6-TCP-degrading activities in JMP134 cell extracts. Our partial purification and initial characterization of the enzyme indicated that a reduced flavin adenine dinucleotide (FADH2)-utilizing monooxygenase converted 2,4,6-TCP to 6-chlorohydroxyquinol (6-CHQ). The finding directed us to PCR amplify a 3.2-kb fragment containing a gene cluster (tcpABC) from JMP134 by using primers designed from conserved regions of FADH2-utilizing monooxygenases and hydroxyquinol 1,2-dioxygenases. Sequence analysis indicated that tcpA, tcpB, and tcpC encoded an FADH2-utilizing monooxygenase, a probable flavin reductase, and a 6-CHQ 1,2-dioxygenase, respectively. The three genes were individually inactivated in JMP134. The tcpA mutant failed to degrade 2,4,6-TCP, while both tcpB and tcpC mutants degraded 2,4,6-TCP to an oxidized product of 6-CHQ. Insertional inactivation of tcpB may have led to a polar effect on downstream tcpC, and this probably resulted in the accumulation of the oxidized form of 6-CHQ. For further characterization, TcpA was produced, purified, and shown to transform 2,4,6-TCP to 6-CHQ when FADH2 was supplied by an Escherichia coli flavin reductase. TcpC produced in E. coli oxidized 6-CHQ to 2-chloromaleylacetate. Thus, our data suggest that JMP134 transforms 2,4,6-TCP to 2-chloromaleylacetate by TcpA and TcpC. Sequence analysis suggests that tcpB may function as an FAD reductase, but experimental data did not support this hypothesis. The function of TcpB remains unknown.
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PMID:Genetic and biochemical characterization of a 2,4,6-trichlorophenol degradation pathway in Ralstonia eutropha JMP134. 1205 43

The tcpRXABCYD operon of Cupriavidus necator JMP134 is involved in the degradation of 2,4,6-trichlorophenol (2,4,6-TCP), a toxic pollutant. TcpA is a reduced flavin adenine dinucleotide (FADH2)-dependent monooxygenase that converts 2,4,6-TCP to 6-chlorohydroxyquinone. It has been implied via genetic analysis that TcpX acts as an FAD reductase to supply TcpA with FADH2, whereas the function of TcpB in 2,4,6-TCP degradation is still unclear. In order to provide direct biochemical evidence for the functions of TcpX and TcpB, the two corresponding genes (tcpX and tcpB) were cloned, overexpressed, and purified in Escherichia coli. TcpX was purified as a C-terminal His tag fusion (TcpX(H)) and found to possess NADH:flavin oxidoreductase activity capable of reducing either FAD or flavin mononucleotide (FMN) with NADH as the reductant. TcpX(H) had no activity toward NADPH or riboflavin. Coupling of TcpX(H) and TcpA demonstrated that TcpX(H) provided FADH2 for TcpA catalysis. Among several substrates tested, TcpB showed the best activity for quinone reduction, with FMN or FAD as the cofactor and NADH as the reductant. TcpB could not replace TcpX(H) in a coupled assay with TcpA for 2,4,6-TCP metabolism, but TcpB could enhance TcpA activity. Further, we showed that TcpB was more effective in reducing 6-chlorohydroxyquinone than chemical reduction alone, using a thiol conjugation assay to probe transitory accumulation of the quinone. Thus, TcpB was acting as a quinone reductase for 6-chlorohydroxyquinone reduction during 2,4,6-TCP degradation.
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PMID:Functions of flavin reductase and quinone reductase in 2,4,6-trichlorophenol degradation by Cupriavidus necator JMP134. 1816 97

Burkholderia cepacia AC1100 completely degrades 2,4,5-trichlorophenol, in which an FADH(2)-dependent monooxygenase (TftD) and an NADH:FAD oxidoreductase (TftC) catalyze the initial steps. TftD oxidizes 2,4,5-trichlorophenol (2,4,5-TCP) to 2,5-dichloro-p-benzoquinone, which is chemically reduced to 2,5-dichloro-p-hydroquinone (2,5-DiCHQ). Then, TftD oxidizes the latter to 5-chloro-2-hydroxy-p-benzoquinone. In those processes, TftC provides all the required FADH(2). We have determined the crystal structures of dimeric TftC and tetrameric TftD at 2.0 and 2.5 A resolution, respectively. The structure of TftC was similar to those of related flavin reductases. The stacked nicotinamide:isoalloxazine rings in TftC and sequential reaction kinetics suggest that the reduced FAD leaves TftC after NADH oxidation. The structure of TftD was also similar to the known structures of FADH(2)-dependent monooxygenases. Its His-289 residue in the re-side of the isoalloxazine ring is within hydrogen bonding distance with a hydroxyl group of 2,5-DiCHQ. An H289A mutation resulted in the complete loss of activity toward 2,5-DiCHQ and a significant decrease in catalytic efficiency toward 2,4,5-TCP. Thus, His-289 plays different roles in the catalysis of 2,4,5-TCP and 2,5-DiCHQ. The results support that free FADH(2) is generated by TftC, and TftD uses FADH(2) to separately transform 2,4,5-TCP and 2,5-DiCHQ. Additional experimental data also support the diffusion of FADH(2) between TftC and TftD without direct physical interaction between the two enzymes.
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PMID:Characterization of chlorophenol 4-monooxygenase (TftD) and NADH:FAD oxidoreductase (TftC) of Burkholderia cepacia AC1100. 1991 6