title: Characterization of an aldolase-dehydrogenase complex that exhibits substrate channeling in the polychlorinated biphenyls degradation pathway.
abstract: An aldolase and dehydrogenase complex from the polychlorinated biphenyl degradation pathway of the bacterium Burkholderia xenovorans LB400 was purified. The aldolase, BphI, had the highest activity with Mn(2+) as the cofactor and was able to transform 4-hydroxy-2-oxopentanoate and 4-hydroxy-2-oxohexanoate to pyruvate and acetaldehyde or propionaldehyde with similar specificity constants. Aldolase activity was competitively inhibited by the pyruvate enolate analogue, oxalate, with a K(ic) of 0.93 microM. The pH-rate profiles suggested the involvement of a pK(a) 7.7 catalytic base in the reaction mechanism. BphI activity was activated 15-fold when substrate turnover was occurring in the dehydrogenase, BphJ, which can be attributed partially to nicotinamide coenzyme binding to BphJ. BphJ had similar specificity constants for acetaldehyde or propionaldehyde and was able to utilize aliphatic aldehydes from two to five carbons in length as substrates, although K(m) values for these aldehyes were >20 mM. When 4-hydroxy-2-oxopentanoate was provided as a substrate to the BphI-BphJ complex in a coupled enzyme assay, no lag in the progress curve of BphJ was observed. When 1 mM propionaldehyde was added exogenously to a reaction mixture containing 0.1 mM 4-hydroxy-2-oxopentanoate, 95% of the CoA esters produced was acetyl CoA. Conversely, 99% of the CoA esters produced was propionyl CoA when a 10-fold molar excess of exogenous acetaldehyde was added in a reaction mixture containing 4-hydroxy-2-oxohexanoate. These results demonstrate that acetaldehyde and propionaldehyde, products of the BphI reaction, are not released in the bulk solvent but are channeled directly to the dehydrogenase.
authors.affiliation: Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada.
authors.forename: Perrin
authors.initials: P
authors.lastname: Baker
authors.forename: Dan
authors.initials: D
authors.lastname: Pan
authors.forename: Jason
authors.initials: J
authors.lastname: Carere
authors.forename: Adam
authors.initials: A
authors.lastname: Rossi
authors.forename: Weijun
authors.initials: W
authors.lastname: Wang
authors.forename: Stephen Y K
authors.initials: SY
authors.lastname: Seah
mesh_terms: D000494:Allosteric Regulation; D001483:Base Sequence; D019117:Burkholderia / Q000201:enzymology; D002384:Catalysis; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D005634:Fructose-Bisphosphate Aldolase / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D010088:Oxidoreductases / Q000378:metabolism*; D011078:Polychlorinated Biphenyls / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D017931:DNA Primers; D011078:Polychlorinated Biphenyls; D010088:Oxidoreductases; D005634:Fructose-Bisphosphate Aldolase