Purification and properties of the physically associated meta-cleavage pathway enzymes 4-hydroxy-2-ketovalerate aldolase and aldehyde dehydrogenase (acylating) from Pseudomonas sp. strain CF600.
The final two steps in the dmp operon-encoded meta-cleavage pathway for phenol degradation in Pseudomonas sp. strain CF600 involve conversion of 4-hydroxy-2-ketovalerate to pyruvate and acetyl coenzyme A (acetyl-CoA) by the enzymes 4-hydroxy-2-ketovalerate aldolase and aldehyde dehydrogenase (acylating) [acetaldehyde:NAD+ oxidoreductase (CoA acetylating), EC 1.2.1.10]. A procedure for purifying these two enzyme activities to homogeneity is reported here. The two activities were found to copurify through five different chromatography steps and ammonium sulfate fractionation, resulting in a preparation that contained approximately equal proportions of two polypeptides with molecular masses of 35 and 40 kDa. Amino-terminal sequencing revealed that the first six amino acids of each polypeptide were those deduced from the previously determined nucleotide sequences of the corresponding dmp operon-encoded genes. The isolated complex had a native molecular mass of 148 kDa, which is consistent with the presence of two of each polypeptide per complex. In addition to generating acetyl-CoA from acetaldehyde, CoA, and NAD+, the dehydrogenase was shown to acylate propionaldehyde, which would be generated by action of the meta-cleavage pathway enzymes on the substrates 3,4-dimethylcatechol and 4-methylcatechol. 4-Hydroxy-2-ketovalerate aldolase activity was stimulated by the addition of Mn2+ and, surprisingly, NADH to assay mixtures. The possible significance of the close physical association between these two polypeptides in ensuring efficient metabolism of the short-chain aldehyde generated by this pathway is discussed.
Powlowski
J
J
Department of Chemistry and Biochemistry, Concordia University, Montreal, Quebec, Canada.
Sahlman
L
L
Shingler
V
V
D000445:Aldehyde Oxidoreductases / Q000302:isolation & purification* / Q000378:metabolism*; D000596:Amino Acids / Q000032:analysis; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D005798:Genes, Bacterial; D007700:Kinetics; D008970:Molecular Weight; D009876:Operon; D007652:Oxo-Acid-Lyases / Q000302:isolation & purification* / Q000378:metabolism*; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics
D000596:Amino Acids; D000445:Aldehyde Oxidoreductases; C027014:acetaldehyde dehydrogenase (acylating); C038482:4-hydroxy-2-ketovalerate aldolase; D007652:Oxo-Acid-Lyases