This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Powlowski, J Articles by Shingler, V Search for Related Content PubMed PubMed Citation Articles by Powlowski, J Articles by Shingler, V

research-article

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.

Department of Chemistry and Biochemistry, Concordia University, Montreal, Quebec, Canada.

ABSTRACT

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.

This article has been cited by other articles:

• Manjasetty, B. A., Powlowski, J., Vrielink, A. (2003). Crystal structure of a bifunctional aldolase-dehydrogenase: Sequestering a reactive and volatile intermediate. Proc. Natl. Acad. Sci. USA 100: 6992-6997 [Abstract] [Full Text]  
• Sakai, M., Miyauchi, K., Kato, N., Masai, E., Fukuda, M. (2003). 2-Hydroxypenta-2,4-dienoate Metabolic Pathway Genes in a Strong Polychlorinated Biphenyl Degrader, Rhodococcus sp. Strain RHA1. Appl. Environ. Microbiol. 69: 427-433 [Abstract] [Full Text]  
• Kretzschmar, U., Schobert, M., Gorisch, H. (2001). The Pseudomonas aeruginosa acsA gene, encoding an acetyl-CoA synthetase, is essential for growth on ethanol. Microbiology 147: 2671-2677 [Abstract] [Full Text]  
• Peng, X., Masai, E., Katayama, Y., Fukuda, M. (1999). Characterization of the meta-Cleavage Compound Hydrolase Gene Involved in Degradation of the Lignin-Related Biphenyl Structure by Sphingomonas paucimobilis SYK-6. Appl. Environ. Microbiol. 65: 2789-2793 [Abstract] [Full Text]  
• Pollard, J. R., Rialland, D., Bugg, T. D. H. (1998). Substrate Selectivity and Biochemical Properties of 4-Hydroxy-2-Keto-Pentanoic Acid Aldolase from Escherichia coli. Appl. Environ. Microbiol. 64: 4093-4094 [Abstract] [Full Text]