Substrate Range of Acetohydroxy Acid Synthase I from Escherichia coli in the Stereoselective Synthesis of A-Hydroxy Ketones Stanislav Engel, 1 Maria Vyazmensky, 2 Dvora Berkovich, 1 Ze’ev Barak, 2 David M. Chipman 2 1 Department of Biotechnology Engineering, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel 2 Department of Life Sciences, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel; telephone: +972-8-647 2646; fax: +972-8-646 1710; e-mail: chipman @bgumail.bgu.ac.il Received 3 June 2004; accepted 16 July 2004 Published online 9 November 2004 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/bit.20275 Abstract: Acetohydroxy acid synthase I appears to be the most effective of the AHAS isozymes found in Escherichia coli in the chiral synthesis of phenylacetyl carbinol from pyruvate and benzaldehyde. We report here the exploration of a range of aldehydes as substrates for AHAS I and dem- onstrate that the enzyme can accept a wide variety of substituted benzaldehydes, as well as heterocyclic and heteroatomic aromatic aldehydes, to produce chiral carbi- nols. The active site of AHAS I does not appear to impose serious steric constraints on the acceptor substrate. The influence of electronic effects on the reaction has been probed using substituted benzaldehydes as substrates. The electrophilicity of the aldehyde acceptor substrates is most important to their reactivity, but the lipophilicity of sub- stituents also affects their reactivity. AHAS I is an effective biosynthetic platform for production of a variety of a- hydroxy ketones, compounds with considerable potential as pharmacological precursors. B 2004 Wiley Periodicals, Inc. Keywords: chiral synthon; acetolactate synthase; aromatic aldehydes; pyruvate; thiamine diphosphate; quantitative structure – activity relationship INTRODUCTION Chiral a-hydroxyketones are versatile building blocks for organic chemistry (Iding et al., 1998). One important chiral a-hydroxyketone is R-phenylacetyl carbinol (R-PAC), used as a synthon in the production of a variety of pharma- ceuticals having a and h adrenergic properties, including l-ephedrine, pseudoephedrine, norephedrine, and phenyl- propanolamine (Rogers et al., 1997; Shukla and Kulkarni, 2000). The manufacture of R-PAC is currently based on a fermentation process in which yeast cells carry out the en- zymatic condensation of pyruvate with externally supplied benzaldehyde (Hildebrandt and Klavehn, 1932; Rogers et al., 1997). The action of yeast pyruvate decarboxylase (PDC) is responsible for this transformation (Hanc and Karac, 1956). The enzyme catalyzes the thiamin diphos- phate (ThDP)-dependent decarboxylation of the a-keto acid pyruvate to acetaldehyde as its physiologically signif- icant reaction but can catalyze condensation (carboligation) of pyruvate with aldehydes to form a-hydroxy ketones as a side reaction (Iding et al., 1998). PDC can use a wide range of aldehydes as acceptor substrates to form a-hydroxy ke- tones (Crout et al., 1994; Iding et al., 1998; Kren et al., 1993; Long et al., 1989), which can be used as precursors for syntheses of new compounds with potential pharmaco- logical activity. Such biotransformations have been carried out using the fermenting yeast Saccharomyces cerevisiae (Long et al., 1989; Schmauder and Groger, 1968), but these are quite inefficient processes due to low reaction rates and low conversion of substrates to the desired products (Long et al., 1989). Formation of a variety of aryl acetyl carbinols using isolated PDC from S. cerevisiae (Kren et al., 1993) or Zymomonas mobilis (Bornemann et al., 1996) has also been reported. The isolated enzymes also have limited efficiency in these biotransformations, suggesting that the intrinsic properties of PDC enzymes are not ideal for this kind of carboligation. We have recently reported the ability of the enzyme acetohydroxy acid synthase (AHAS or acetolactate syn- thase, EC 4.1.3.18) to catalyze the enantiospecific con- densation of benzaldehyde with pyruvate to form R-PAC (Engel et al., 2003). This enzyme, like pyruvate decarbox- ylase, belongs to a homologous family of ThDP-dependent enzymes whose normal reaction begins with decarboxyla- tion of pyruvate (Fig. 1, step 1) (Bowen et al., 1997; Green, 1989). This step provides the thermodynamic driving force B 2004 Wiley Periodicals, Inc. Correspondence to: David M. Chipman Contract grant sponsor: BG Negev Technologies