Origin of the specificities of acetohydroxyacid synthases and glyoxylate carboligase

David M. Chipman, Ze'ev Barak, Boaz Shaanan, Maria Vyazmensky, Elad Binshtein, Inna Belenky, Vered Temam, Andrea Steinmetz, Ralph Golbik, Kai Tittmann

Research output: Contribution to journalArticlepeer-review

12 Scopus citations


Acetohydroxyacid synthases (AHAS) and glyoxylate carboligase (GCL) catalyze decarboxylation of 2-ketoacids and condensation of the resulting hydroxyalkylThDP anions/enamines with a second ketoacid to form 2-acyl-2-hydroxyacids. AHASs prefer pyruvate by >10-fold over any other ketoacid as first substrate. Steric hindrance seems to be the major determinant of this specificity; Escherichia coli AHAS isozyme II mutant Val375Ala allows 2-ketobutyrate (C2H5COCO2-) to be a good first substrate and the mutant enzyme can thus synthesize 2-propio-2-hydroxybutyrate. An Ile residue in the equivalent position in GCL (Ile393) may play the analogous role in restricting GCL to glyoxylate (HCOCO2-) as first substrate. The specificity of AHAS for 2-ketoacids as acceptor substrates is due to an arginine residue which probably interacts with the carboxylate of the second substrate (e.g., Arg276 in AHASII). Mutants altered at this arginine can utilize aromatic aldehydes as second substrate and form chiral arylacyl carbinols, of interest as precursors for pharmaceutical syntheses. Analysis of AHAS II supports a mechanism in which carboligation occurs after rate-determining formation of hydroxyethylThDP. NMR measurements of the distribution of ThDP-bound intermediates showed that a faster rate constant for product release when the alkyl group derived from the acceptor substrate is ethyl compared to methyl plays a major role in product specificity. The crucial role of a Trp residue (Trp 464 in AHASII) in determining specificity may be due to control of a conformational change involved in product release rather than to affinity for 2-ketobutyrate. It is significant that in AHAS I, without the required Trp and with a low specificity for 2-ketobutyrate as acceptor substrate, the product release step is rapidly reversible.

Original languageEnglish
Pages (from-to)50-55
Number of pages6
JournalJournal of Molecular Catalysis - B Enzymatic
Issue number1-2
StatePublished - 1 Nov 2009


  • Acetohydroxy acid synthase
  • Carboligase
  • Glyoxylate carboligase
  • Specificity
  • Thiamin diphosphate

ASJC Scopus subject areas

  • Catalysis
  • Bioengineering
  • Biochemistry
  • Process Chemistry and Technology


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