nag Genes of Ralstonia (Formerly Pseudomonas ) sp. Strain U2 Encoding Enzymes for Gentisate Catabolism

Abstract

ABSTRACT Ralstonia sp. strain U2 metabolizes naphthalene via gentisate to central metabolites. We have cloned and sequenced a 21.6-kb region spanning the nag genes. Upstream of the pathway genes are nagY, homologous to chemotaxis proteins, and nagR, a regulatory gene of the LysR family. Divergently transcribed from nagR are the genes for conversion of naphthalene to gentisate ( nagAaGHAbAcAdBFCQED ) (S. L. Fuenmayor, M. Wild, A. L. Boyes, and P. A. Williams, J. Bacteriol. 180:2522–2530, 1998), which except for the insertion of nagGH , encoding the salicylate 5-hydroxylase, are homologous to and in the same order as the genes in the classical upper pathway operon described for conversion of naphthalene to salicylate found in the NAH7 plasmid of Pseudomonas putida PpG7. Downstream of nahD is a cluster of genes ( nagJIKLMN ) which are probably cotranscribed with nagAaGHAbAcAdBFCQED as a single large operon. By cloning into expression vectors and by biochemical assays, three of these genes ( nagIKL ) have been shown to encode the enzymes involved in the further catabolism of gentisate to fumarate and pyruvate. NagI is a gentisate 1,2-dioxygenase which converts gentisate to maleylpyruvate and is also able to catalyze the oxidation of some substituted gentisates. NagL is a reduced glutathione-dependent maleylpyruvate isomerase catalyzing the isomerization of maleylpyruvate to fumarylpyruvate. NagK is a fumarylpyruvate hydrolase which hydrolyzes fumarylpyruvate to fumarate and pyruvate. The three other genes ( nagJMN ) have also been cloned and overexpressed, but no biochemical activities have been attributed to them. NagJ is homologous to a glutathione S -transferase, and NagM and NagN are proteins homologous to each other and to other proteins of unknown function. Downstream of the operon is a partial sequence with homology to a transposase.