Purification, characterisation and reaction mechanism of monofunctional 2-hydroxypentadienoic acid hydratase from Escherichia coli.
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.
Pollard
J R
JR
Department of Chemistry, University of Southampton, Highfield, UK.
Bugg
T D
TD
D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000201:enzymology*; D005231:Fatty Acids, Unsaturated / Q000737:chemistry / Q000378:metabolism; D006836:Hydro-Lyases / Q000302:isolation & purification* / Q000378:metabolism*; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid
D005231:Fatty Acids, Unsaturated; C061660:2-hydroxy-2,4-pentadienoic acid; C110907:2-hydroxypentadienoic acid hydratase; D006836:Hydro-Lyases