Molecular and enzymatic analysis of the "aldoxime-nitrile pathway" in the glutaronitrile degrader Pseudomonas sp. K-9.
A gene cluster responsible for aldoxime metabolism in the glutaronitrile degrader Pseudomonas sp. K-9 was analyzed genetically and enzymatically. The cluster was composed of genes coding for aldoxime dehydratase (Oxd), nitrile hydratase (NHase), NHase activator, amidase, acyl-CoA ligase, and some regulatory and functionally unknown proteins, which were similar to proteins appearing in the "aldoxime-nitrile pathway" gene cluster from strains having Fe-containing NHase. A key enzyme in the cluster, OxdK, which has 32.7-90.3 % identity with known Oxds, was overexpressed in Escherichia coli cells under the control of a T7 promoter in its His(6)-tagged form, purified, and characterized. The enzyme showed similar characteristics with the known Oxds coexisting with an Fe-containing NHase in its subunit structure, substrate specificity, and effects on various compounds. The enzyme can be classified into a group of "aliphatic aldoxime dehydratase (EC 4.99.1.5)." The existence of a gene cluster of enzymes responsible for aldoxime metabolism via the aldoxime-nitrile pathway (aldoxime-->nitrile-->amide-->acid-->acyl-CoA) in Pseudomonas sp. K-9, and the fact that the proteins comprising the cluster are similar to those acting on aliphatic type substrates, evidently clarified the alkylaldoxime-degrading pathway in that strain.
Biotechnology Research Center, Faculty of Engineering, Toyama Prefectural University, 5180 Kurokawa, Kosugi, Toyama 939-0398, Japan.
Yasuo
Y
Kato
Yasuhisa
Y
Asano
D000619:Aminohydrolases / Q000378:metabolism; D001426:Bacterial Proteins; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D006836:Hydro-Lyases / Q000378:metabolism; D005810:Multigene Family; D009570:Nitriles / Q000378:metabolism*; D010091:Oximes / Q000378:metabolism*; D011549:Pseudomonas / Q000145:classification / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D009570:Nitriles; D010091:Oximes; D000619:Aminohydrolases; D006836:Hydro-Lyases; C412124:aldoxime dehydratase