title: Comparison of two metal-dependent pyruvate aldolases related by convergent evolution: substrate specificity, kinetic mechanism, and substrate channeling.
abstract: HpaI and BphI are two pyruvate class II aldolases found in aromatic meta-cleavage degradation pathways that catalyze similar reactions but are not related in sequence. Steady-state kinetic analysis of the aldol addition reactions and product inhibition assays showed that HpaI exhibits a rapid equilibrium random order mechanism while BphI exhibits a compulsory order mechanism, with pyruvate binding first. Both aldolases are able to utilize aldehyde acceptors two to five carbons in length; however, HpaI showed broader specificity and had a preference for aldehydes containing longer linear alkyl chains or C2-OH substitutions. Both enzymes were able to bind 2-keto acids larger than pyruvate, but only HpaI was able to utilize both pyruvate and 2-ketobutanoate as carbonyl donors in the aldol addition reaction. HpaI lacks stereospecific control producing racemic mixtures of 4-hydroxy-2-oxopentanoate (HOPA) from pyruvate and acetaldehyde while BphI synthesizes only (4S)-HOPA. BphI is also able to utilize acetaldehyde produced by the reduction of acetyl-CoA catalyzed by the associated aldehyde dehydrogenase, BphJ. This aldehyde was directly channeled from the dehydrogenase to the aldolase active sites, with an efficiency of 84%. Furthermore, the BphJ reductive deacylation reaction increased 4-fold when BphI was catalyzing the aldol addition reaction. Therefore, the BphI-BphJ enzyme complex exhibits unique bidirectionality in substrate channeling and allosteric activation.
authors.affiliation: Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada N1G 2W1.
authors.forename: Weijun
authors.initials: W
authors.lastname: Wang
authors.forename: Perrin
authors.initials: P
authors.lastname: Baker
authors.forename: Stephen Y K
authors.initials: SY
authors.lastname: Seah
mesh_terms: D000444:Aldehyde Dehydrogenase / Q000378:metabolism*; D000446:Aldehyde-Lyases / Q000378:metabolism*; D000447:Aldehydes / Q000378:metabolism*; D001665:Binding Sites; D002384:Catalysis; D020134:Catalytic Domain; D029968:Escherichia coli Proteins / Q000378:metabolism*; D019143:Evolution, Molecular; D007700:Kinetics; D011487:Protein Conformation; D011773:Pyruvates / Q000378:metabolism*; D013237:Stereoisomerism; D013379:Substrate Specificity
chemical_list: D000447:Aldehydes; D029968:Escherichia coli Proteins; D011773:Pyruvates; D000444:Aldehyde Dehydrogenase; D000446:Aldehyde-Lyases; C503556:HpaI protein, E coli