Comparison of two metal-dependent pyruvate aldolases related by convergent evolution: substrate specificity, kinetic mechanism, and substrate channeling

Biochemistry. 2010 May 4;49(17):3774-82. doi: 10.1021/bi100251u.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aldehyde Dehydrogenase / metabolism*
  • Aldehyde-Lyases / metabolism*
  • Aldehydes / metabolism*
  • Binding Sites
  • Catalysis
  • Catalytic Domain
  • Escherichia coli Proteins / metabolism*
  • Evolution, Molecular*
  • Kinetics
  • Protein Conformation
  • Pyruvates / metabolism*
  • Stereoisomerism
  • Substrate Specificity


  • Aldehydes
  • Escherichia coli Proteins
  • Pyruvates
  • Aldehyde Dehydrogenase
  • Aldehyde-Lyases
  • HpaI protein, E coli