Cloning, sequencing, and analysis of a gene cluster from Chelatobacter heintzii ATCC 29600 encoding nitrilotriacetate monooxygenase and NADH:flavin mononucleotide oxidoreductase.
Nitrilotriacetate (NTA) is an important chelating agent in detergents and has also been used extensively in processing radionuclides. In Chelatobacter heintzii ATCC 29600, biodegradation of NTA is initiated by NTA monooxygenase that oxidizes NTA to iminodiacetate and glyoxylate. The NTA monooxygenase activity requires two component proteins, component A and component B, but the function of each component is unclear. We have cloned and sequenced a gene cluster encoding components A and B (nmoA and nmoB) and two additional open reading frames, nmoR and nmoT, downstream of nmoA. Based on sequence similarities, nmoR and nmoT probably encode a regulatory protein and a transposase, respectively. The NmoA sequence was similar to a monooxygenase that uses reduced flavin mononucleotide (FMNH2) as reductant; NmoB was similar to an NADH:flavin mononucleotide (FMN) oxidoreductase. On the basis of this information, we tested the function of each component. Purified component B was shown to be an NADH:FMN oxidoreductase, and its activity could be separated from that of component A. When the Photobacterium fischeri NADH:FMN oxidoreductase was substituted for component B in the complete reaction, NTA was oxidized, showing that the substrate specificity of the reaction resides in component A. Component A is therefore an NTA monooxygenase that uses FMNH2 and O2 to oxidize NTA, and component B is an NADH:FMN oxidoreductase that provides FMNH2 for NTA oxidation.
Xu
Y
Y
Department of Microbiology, Washington State University Tri-Cities, Richland 99352, USA.
Mortimer
M W
MW
Fisher
T S
TS
Kahn
M L
ML
Brockman
F J
FJ
Xun
L
L
D000595:Amino Acid Sequence; D001483:Base Sequence; D001673:Biodegradation, Environmental; D003001:Cloning, Molecular; D038181:FMN Reductase; D005486:Flavin Mononucleotide / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D006088:Gram-Negative Aerobic Bacteria / Q000201:enzymology / Q000235:genetics*; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D005810:Multigene Family; D009243:NAD / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D009571:Nitrilotriacetic Acid / Q000378:metabolism; D016366:Open Reading Frames; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment
D011994:Recombinant Proteins; D009243:NAD; D005486:Flavin Mononucleotide; D006899:Mixed Function Oxygenases; C072680:nitrilotriacetate monooxygenase; D038181:FMN Reductase; D009247:NADH, NADPH Oxidoreductases; D009571:Nitrilotriacetic Acid