Recent Papers

Zhou N-Y.,  Al-Dulayyami J., Baird M.S.  &  Williams P.A. (2002)  Salicylate 5-hydroxylase from Ralstonia sp. strain U2: a monooxygenase with close relationships to and shared electron transport proteins with naphthalene dioxygenase. J.Bacteriol. 184: (in press).

ABSTRACT

The genes from the oxygenase cluster nagAaGHAbAcAd  of naphthalene-degrading Ralstonia sp. strain U2 were cloned and overexpressed. Salicylate 5-hydroxylase (S5H) activity, converting salicylate to gentisate, was present in vitro only in the single extract of cells with over-expressed nagAaGHAb or in a mixture of three cell extracts containing respectively NagGH (the oxygenase components),  NagAa (ferredoxin reductase) and NagAb (ferredoxin). Each of the three extracts required for S5H activity was rate-limiting in the presence of excess of the others but, when in excess, did not affect the rate of catalysis. S5H catalysed the 5-hydroxylation of the aromatic rings of 3- and 4-substituted salicylates. However the methyl group of 5-methylsalicylate was hydroxylated to produce the 5-hydroxymethyl derivative and the 6-position on the ring of 5-chlorosalicylate was hydroxylated producing 5-chloro-2,6-dihydroxybenzoate. In an assay for the nag naphthalene dioxygenase (NDO) based on the indole-linked oxidation of NADH, three extracts were essential for activity (NagAcAd, NagAa, NagAb). NDO and S5H were assayed in the presence of all possible combinations of the nag proteins and the corresponding nah NDO proteins from the 'classical' naphthalene degrader P. putida NCIMB9816. All three oxygenase components functioned with mixed combinations of the electron transport proteins from either strain. The S5H from strain U2 is a unique monooxygenase which shares both sequence similarity with dioxygenases such as NDO but is also sufficiently similar in structure to interact with the same electron transport chain and probably does so in vivo during naphthalene catabolism in strain U2.