Ning-yi ZHOU, Sergio L. FUENMAYOR, and Peter A. WILLIAMS. nag
Genes of Ralstonia (formerly Pseudomonas) sp. strain U2 encoding
the enzymes for gentisate catabolism. J.Bacteriol.
183
(2001) 700-708.
ABSTRACT
Ralstonia sp. strain U2 metabolises 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) 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 on 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 (GDO) which converts gentisate to maleylpyruvate
and is also able to catalyse the oxidation of some substituted gentisates.
NagL is a reduced glutathione (GSH)-dependent maleylpyruvate isomerase
catalyzing the isomerisation of maleylpyruvate to fumarylpyruvate. NagK
is a fumarylpyruvate hydrolase which hydrolyses fumarylpyruvate to fumarate
and pyruvate. The three other genes (
nagJMN) have also been cloned
and over-expressed 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.
