Salicylate 5-Hydroxylase from Ralstonia sp. Strain U2: a Monooxygenase with Close Relationships to and Shared Electron Transport Proteins with Naphthalene Dioxygenase

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 overexpressed 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 catalyzed 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, and 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 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.The classical pathway for naphthalene catabolism in bacteria such as Pseudomonas putida strains NCIMB9816 and PpG7 (7, 52, 53) is via dihydroxylation and cleavage of the first ring and removal of the resulting aliphatic side chain to produce salicylate (2-hydroxybenzoate). This is then converted by the action of salicylate 1-hydroxylase to catechol (1,2-dihydroxybenzene), which undergoes extradiol cleavage via the same route used for a wide range of other aromatic compounds such as toluene and the xylenes (16) and phenol (38). The nah genes are located on two separate operons: the upper pathway operon encoding the conversion of naphthalene to salicylate and the lower (or meta) pathway operon encoding the conversion of salicylate to acetyl coenzyme A and pyruvate (5, 52). The induction of the two operons is linked by a common regulator protein, NahR (34).Although a route for naphthalene catabolism involving the alternative conversion of salicylate to gentisate (2,5-dihydroxybenzoate) has been known for some time (14,25,30,39,41,53), genetic analysis of such a pathway, in Ralstonia (formerly Pseudomonas) sp. strain U2, has only recently been reported (12, 54). There is similarity between the nag genes of strain U2 and the classical nah genes, but only in the conversion of naphthalene to salicylate, for which the genes are homologous and in the same ord...

A Large Gene Cluster in Burkholderia xenovorans Encoding Abietane Diterpenoid Catabolism

“…A common electron transport system, shared by both monooxygenase and dioxygenase systems, was previously demonstrated in the catabolic pathway for naphthalene in Ralstonia sp. strain U2 (40). FIG.…”

The Naphthalene Catabolic ( nag ) Genes of Ralstonia sp. Strain U2 Are an Operon That Is Regulated by NagR, a LysR-Type Transcriptional Regulator

“…In all of these cases, the four genes (AaAbAcAd) encoding the nitroaromatic dioxygenase are homologs of naphthalene dioxygenase genes, but the sequences are more similar to the sequences of the nag genes than to the sequences of the nah genes (8,12,29). Additionally, between the Aa and Ab genes they all contain residual sequences homologous to nagGH, the genes encoding salicylate 5-hydroxylase (8,29).…”

“…In all of these cases, the four genes (AaAbAcAd) encoding the nitroaromatic dioxygenase are homologs of naphthalene dioxygenase genes, but the sequences are more similar to the sequences of the nag genes than to the sequences of the nah genes (8,12,29). Additionally, between the Aa and Ab genes they all contain residual sequences homologous to nagGH, the genes encoding salicylate 5-hydroxylase (8,29). This fact has been interpreted as showing that the nitroaromatic dioxygenases evolved by recruitment of nag-like naphthalene dioxygenase genes carrying the nagGH insert but subsequently acquired inactivating mutations in the nagGH DNA but the nagGH DNA was not completely deleted (12,18,29).…”

“…strain U2 code for the enzymes of the alternative pathway for the catabolism of naphthalene, which converts naphthalene to fumarate and pyruvate via salicylate (2-hydroxybenzoate) and gentisate (2,5-dihydroxybenzoate), and they are organized in a continuous sequence of adjacent genes (8,28,29). This is in contrast to the classical naphthalene pathway enzymes, which are encoded on two separate operons, the upper-pathway operon (genes nahAa to nahF) for the conversion of naphthalene to salicylate and the lower-pathway (or meta-pathway) operon (genes nahG to nahM) for the conversion of salicylate to acetyl coenzyme A and pyruvate via catechol (5,27).…”

Molecular and Biochemical Characterization of the xlnD -Encoded 3-Hydroxybenzoate 6-Hydroxylase Involved in the Degradation of 2,5-Xylenol via the Gentisate Pathway in Pseudomonas alcaligenes NCIMB 9867

“…The sample cuvette contained 100 mM potassium phosphate buffer, 0.2 mol of substrate, and 0.5 mol of NADH. The problem of high endogenous NADH oxidase activity, present in crude cell extracts of P. alcaligenes and the E. coli host but not in the purified 6-hydroxylase, was overcome by using the reference blank cuvette containing all assay components except the substrate to compensate for NADH oxidase, similar to the spectrophotometric assay used to detect salicylate 5-hydroxylase activity (30). The assay was initiated by the addition of ultracentrifuged extracts to both sample and reference cuvettes.…”