Publications

• Purification, characterisation and reaction mechanism of monofunctional 2-hydroxypentadienoic acid hydratase from Escherichia coli.

  Pollard J.R., Bugg T.D.H.

  2-Hydroxypentadienoic acid hydratase is found on many bacterial catabolic pathways responsible for the degradation of aromatic compounds. Monofunctional 2-hydroxypentadienoic acid hydratase from Escherichia coli has been purified 3800-fold to homogeneity, using enzymatically generated 2-hydroxypen ... >> More

  2-Hydroxypentadienoic acid hydratase is found on many bacterial catabolic pathways responsible for the degradation of aromatic compounds. Monofunctional 2-hydroxypentadienoic acid hydratase from Escherichia coli has been purified 3800-fold to homogeneity, using enzymatically generated 2-hydroxypentadienoic acid as substrate. The purified 28-kDa protein requires a divalent metal ion for activity, optimum activity being obtained with Mn2+. Steady-state kinetic parameters were measured (Km = 41 +/-4 microM, k(cat) = 450 s(-1)), the enzyme exhibiting substrate inhibition at high substrate concentrations. The pH/rate profile and inhibition by group-specific reagents were examined, and evidence was obtained for essential cysteine and tryptophan residues. An amino acid sequence alignment of the inferred amino acid sequence with nine other sequences was carried out and revealed several conserved sequence motifs. The substrate for the enzymatic reaction was found to be the dienol tautomer of 2-hydroxypentadienoic acid. Analysis of the reaction products by HPLC confirmed the identity of the 4-hydroxy-2-ketopentanoic acid product. Analogues of possible reaction intermediates were tested as inhibitors, and sodium oxalate was found to act as a potent enzyme inhibitor (Ki = 4.9 +/-0.7 microM). The potent inhibition by oxalate is consistent with a mechanism in which tautomerisation to 2-ketopent-3-enoic acid takes place at the active site, followed by conjugate addition of water. << Less

  Eur. J. Biochem. 251:98-106(1998) [PubMed] [EuropePMC]

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

  Kunz D.A., Ribbons D.W., Chapman P.J.

  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 ... >> More

  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. << Less

  J Bacteriol 148:72-82(1981) [PubMed] [EuropePMC]