title: Investigating the molecular determinants for substrate channeling in BphI-BphJ, an aldolase-dehydrogenase complex from the polychlorinated biphenyls degradation pathway.
abstract: BphI-BphJ, an aldolase-dehydrogenase complex from the polychlorinated biphenyls (PCBs) degradation pathway, cleaves 4-hydroxy-2-oxoacids to pyruvate and an aldehyde. The enzyme complex was shown to exhibit substrate channeling, whereby linear aldehydes of up to 6 carbons long and branched isobutyraldehyde were directly channeled from the aldolase to the dehydrogenase with greater than 80% efficiency. BphI variants G322F, G322L, and G323F were created and were found to block aldehyde channeling. The dehydrogenase cofactor NADH was able to activate the catalytic activity of the aldol cleavage reaction in these variants, suggesting that activation of BphI by BphJ cofactors is not solely due to faster aldehyde release. A G323L variant was able to channel acetaldehyde but not the larger propionaldehyde while the G323A variant was able to channel butyraldehyde but not its isomer isobutyraldehyde, confirming that the restricted channeling of aldehydes in these glycine variants are due to steric blockage of the channel. Substitution of His-20 and Tyr-290 in BphI led to significant reductions in aldehyde channeling efficiencies. A mechanism of substrate channeling involving these two gating residues is proposed.
authors.affiliation: Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada N1G 2W1.
authors.forename: Jason
authors.initials: J
authors.lastname: Carere
authors.forename: Perrin
authors.initials: P
authors.lastname: Baker
authors.forename: Stephen Y K
authors.initials: SY
authors.lastname: Seah
mesh_terms: D000445:Aldehyde Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D000446:Aldehyde-Lyases / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011078:Polychlorinated Biphenyls / Q000378:metabolism*; D016415:Sequence Alignment; D013379:Substrate Specificity
chemical_list: D011078:Polychlorinated Biphenyls; D000445:Aldehyde Oxidoreductases; D000446:Aldehyde-Lyases