2-hydroxymuconic Semialdehyde Dehydrogenase Research Articles

Metabolism of allylglycine and cis-crotylglycine by Pseudomonas putida (arvilla) mt-2 harboring a TOL plasmid.

Spontaneous mutants which acquired the ability to utilize d-allylglycine (d-2-amino-4-pentenoic acid) and dl-cis-crotylglycine (dl-2-amino-cis-4-hexenoic acid) but not l-allylglycine or dl-trans-crotylglycine could be readily isolated from Pseudomonas putida mt-2 (PaM1). Derivative strains of PaM1 putatively cured of the TOL (pWWO) plasmid were incapable of forming mutants able to utilize the amino acids for growth; however, this ability could be regained by conjugative transfer of the TOL (pWWO) plasmid from a wild-type strain of mt-2 or of the TOL (pDK1) plasmid from a related strain of P. putida (HS1), into cured recipients. dl-Allylglycine-grown cells of one spontaneous mutant (PaM1000) extensively oxidized dl-allylglycine and dl-cis-crotylglycine, whereas only a limited oxidation was observed toward l-allylglycine and dl-trans-crotylglycine. Cell extracts prepared from PaM1000 cells contained high levels of 2-keto-4-hydroxyvalerate aldolase and 2-keto-4-pentenoic acid hydratase, the latter enzyme showing higher activity toward 2-keto-cis-4-hexenoic acid than toward the trans isomer. Levels of other enzymes of the TOL degradative pathway, including toluate oxidase, catechol-2,3-oxygenase, 2-hydroxymuconic semialdehyde hydrolase, and 2-hydroxymuconic semialdehyde dehydrogenase, were also found to be elevated after growth on allylglycine. Whole cells of a putative cured strain, PaM3, accumulated 2-keto-4-pentenoic acid from d-allylglycine, which was shown to be rapidly degraded by cell extracts of PaM1000 grown on dl-allylglycine. These same cell extracts were also capable of catalyzing the dehydrogenation of d- but not l-allylglycine and were further found to metabolize the amino acid completely to pyruvate and acetaldehyde. Differential centrifugation of crude cell extracts localized d-allylglycine dehydrogenase activity to membrane fractions. The results are consistent with a catabolic pathway for d-allylglycine and dl-cis-crotylglycine involving the corresponding keto-enoic acids as intermediates, the further metabolism of which is effected by the action of TOL plasmid-encoded enzymes.

Physical and functional mapping of RP4-TOL plasmid recombinants: analysis of insertion and deletion mutants.

Cleavage sites for the restriction endonucleases XhoI, BamHI, HindIII, and EcoRI were mapped on the pTN2 plasmid, a recombinant of TOL and RP4, which specifies the toluene-degrading enzymes in the same way as the wild-type TOL plasmid. The pTN2 plasmid, purified from a strain of Escherichia coli, contained the entire length of the RP4 plasmid (about 54 kilobase pairs [kb]) and the TOL segment (about 56 kb). The TOL segment is inserted at about 12 and 5 kb away from the EcoRI and BamHI cleavage sites of RP4, respectively. Cleavage sites for XhoI, BamHI, HindIII, and EcoRI were also mapped on an insertion mutant, pTN1, and two deletion mutants, pTN81 and pTN9. Analysis of pTN81 and pTN9 allowed estimation of the region of the gene cluster for benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase, as well as the region required for toluate oxygenase activity. Induction of TOL enzymes directed by pTN1 suggested the location and orientation of transcription of the gene cluster for catechol 2,3-oxygenase, 2-hydroxymuconic semialdehyde dehydrogenase, and 2-hydroxymuconic semialdehyde hydrolase. Analysis of strains carrying both pTN9 and a xylR mutant of the TOL plasmid demonstrated that xylR+ is trans dominant over xylR.

Regulation of the Plasmid-specified Naphthalene Catabolic Pathway of Pseudomonas putida

The regulation of the catabolic pathway for the degradation of naphthalene specified by the plasmids NAH, pND140 and pND160 was studied using plasmid-borne regulatory mutants in a Pseudomonas putida host strain. Growth of strains harbouring the parent plasmids in the presence of salicylate resulted in induction of selected enzymes involved in the conversion of naphthalene to catechol (naphthalene oxygenase, salicylaldehyde dehydrogenase and salicylate hydroxylase) and enzymes of the meta-cleavage pathway (catechol 2,3-dioxygenase, 2-hydroxymuconic semialdehyde hydrolase and 2-hydroxymuconic semi-aldehyde dehydrogenase). Partial induction was also observed for all the NAH-encoded enzymes assayed when using m-toluate as the inducing compound, over a genetic block. Mutants were obtained for each plasmid where the three enzymes of the meta-cleavage pathway were produced constitutively suggesting that the enzymes of the meta-cleavage pathway belong to one operon. In these mutants, enzymes involved in the conversion of naphthalene to catechol were not produced constitutively but remained inducible during growth on salicylate indicating that these enzymes belong to a separate operon or operons.

A Partial Order for Genes Determining Enzymes of the meta-Cleavage Pathway in Pseudomonas putida

SUMMARY: A partial order for genes which specify meta-cleavage pathway enzymes has been derived from properties of a polarity mutant strain (PsU5) of Pseudomonas putida ncib10015 and a partial revertant, PsU5/R21. The polar mutation is within the 2-hydroxymuconic semialdehyde dehydrogenase gene and results in loss of detectable 2-hydroxymuconic semialdehyde hydrolase and 90 to 95 % reduction in the activities of 4-oxalocrotonate tautomerase, 4-oxalocrotonate decarboxylase, 2-oxopent-4-enoate hydratase and 4-hydroxy-2-oxovalerate aldolase. The partial revertant PsU5/R21 regained wild-type levels of all enzymes except the aldehyde dehydrogenase. It is probable that all genes determining the dehydrogenase and subsequent enzymes are transcribed as a polycistronic message. This is the first report of mapping genes determining the enzymes of the meta-cleavage pathway.

Pathways for the degradation of m-cresol and p-cresol by Pseudomonas putida.

A comparison of the oxidation rates of various compounds by whole cells of Pseudomonas putida 3, 5 indicated that m-cresol is metabolized by oxidation to 3-hydroxybenzoate followed by hydroxylation to gentisate, the ring-fission substrate, when grown with 3, 5-xylenol. However, when m-cresol was the growth substrate, similar experiments suggested a different pathway involving a methyl-substituted catechol, and ring-fission by meta cleavage. Assays of ring-fission enzymes in cell-free extracts confirmed that different pathways are induced by the two growth substrates. 3, 5-Xylenol-grown cells contained high levels of gentisate oxygenase and only very small amounts of catechol oxygenase, whereas gentisate ocygenase could not be detected in m-cresol-grown cells, but levels of catechol oxygenase were greatly increased. Extracts of m-cresol-grown cells also contained 2-hydroxymuconic semialdehyde dehydrogenase and hydrolase, whose specificities enable them to metabolize the ring-fission products from catechol, 3-methylcatechol, and 4-methylcatechol. This catechol pathway is also used by m-cresol-grown cells for p-cresol metabolism. In contrast, the results for cells grown with p-cresol point to an alternative pathway involving oxidation to 4-hydroxybenzoate and hydrosylation to protocatechuate as ring-fission substrate. Extracts of these cells contained high levels of protocatechuate oxygenase and only small amounts of catechol oxygenase.

The metabolic divergence in the meta cleavage of catechols by Pseudomonas putida NCIB 10015. Physiological significance and evolutionary implications.

A reinvestigation of the catabolic pathway(s) used by Pseudomonas putida NCIB 10015 (Dagley's strain) for the degradation of phenol and the cresols has proved the existence of a metabolic divergence after meta cleavage of the catechols formed by hydroxylation of the primary substrates. The ring‐fission products of catechol and 4‐methylcatechol are shown to be simultaneously catabolized by two different enzymic activities, an NAD+‐dependent dehydrogenase and a cofactor‐independent hydrolase. The metabolizing activities of both ring‐fission products in extracts of cells grown on phenol and the cresols (o‐, m‐ and p‐cresol) were found to be non specific; thermal inactivation of extracts of phenol‐grown cells has shown that this nonspecificity is attributable to only one enzyme expressing each activity and that the two activities are located on separate proteins. Extracts of cells grown on all four substrates contain high induced levels of the meta cleavage suite of enzymes functional in the dissimilation of catechol, including both the 4‐oxalocrotonate branch (NAD+‐dependent 2‐hydroxymuconic semialdehyde dehydrogenase, 4‐oxalocrotonate tautomerase and 4‐oxalocrotonate decarboxylase) and the hydrolytic branch (2‐hydroxymuconic semialdehyde hydrolase). The hydroxylase, oxygenase, dehydrogenase and hydrolase activities are shown to be nonspecific and can also act upon the methyl derivatives of their respective substrates. A constant pattern of specificity was found for these enzymes, independent of the monophenolic substrate used for growth. From studies with a mutant lacking phenol hydroxylase, the entire suite of meta cleavage enzymes are shown to be coincidently induced from the top by the primary substrate (phenol or the cresols). The evolutionary and physiological implications of the divergent pathway are discussed.