The thiamin diphosphate (ThDP)-dependent biosynthetic enzyme acetohydroxyacid synthase (AHAS) catalyzes decarboxylation of pyruvate and specific condensation of the resulting ThDP-bound two-carbon intermediate, hydroxyethyl-ThDP anion/enamine (HEThDP ؊ ), with a second ketoacid, to form acetolactate or acetohydroxybutyrate. Whereas the mechanism of formation of HEThDP ؊ from pyruvate is well understood, the role of the enzyme in control of the carboligation reaction of HEThDP ؊ is not. Recent crystal structures of yeast AHAS from Duggleby's laboratory suggested that an arginine residue might interact with the second ketoacid substrate. Mutagenesis of this completely conserved residue in Escherichia coli AHAS isozyme II (Arg 276 ) confirms that it is required for rapid and specific reaction of the second ketoacid. In the mutant proteins, the normally rapid second phase of the reaction becomes rate-determining. A competing alternative nonnatural but stereospecific reaction of bound Acetohydroxyacid synthase (AHAS) 1 belongs to a homologous family of thiamin diphosphate (ThDP)-dependent enzymes that catalyze reactions whose initial step is decarboxylation of pyruvate or another 2-ketoacid (1, 2). However, despite the similarity of AHASs to, for example, pyruvate decarboxylases and pyruvate oxidases (3-6), AHASs carry out a specific carboligation reaction in which the decarboxylation of pyruvate is followed by the condensation of the bound hydroxyethyl-ThDP anion/enamine (HEThDP Ϫ ) intermediate with a second aliphatic ketoacid to form an acetohydroxyacid (Fig. 1). Whereas the role of the enzyme in the first steps in AHAS catalysis (i.e. activation of ThDP (7), decarboxylation of pyruvate, and formation of HEThDP Ϫ (step 1 in Fig. 1)) is comparable with the function of other members of its homologous family (8), it has been difficult to suggest roles for specific protein residues in the final steps (2 and 3) of the reaction in which the product acetohydroxyacid is formed and released.One reason for this uncertainty has been the lack of clear direct information on the structure of the regions of the active site that might be involved in selective reaction of HEThDP Ϫ with a second ketoacid. Although we have proposed a homology model for AHAS isozyme II from Escherichia coli (9), based on the crystal structure of pyruvate oxidase from Lactobacillus plantarum (LpPOX) (4), these two proteins have very different sequences in the region that is likely to interact with the second substrate. In the first published crystal structure of an AHAS, that of the catalytic subunits of the yeast enzyme (10), this region was disordered. The recent publication of a new structure of the yeast enzyme with a tightly bound herbicide (11) now provides a solid framework for consideration of the role of the protein in directing the fate of the HEThDP Ϫ intermediate. A second, equally serious obstacle to the understanding of the mechanism of AHAS has been a lack of experimental tools for studying the rates of individual steps in the reaction ...
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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 demonstrate that the enzyme can accept a wide variety of substituted benzaldehydes, as well as heterocyclic and heteroatomic aromatic aldehydes, to produce chiral carbinols. 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 substituents also affects their reactivity. AHAS I is an effective biosynthetic platform for production of a variety of alpha-hydroxy ketones, compounds with considerable potential as pharmacological precursors.
We tested the possibility of utilizing acetohydroxyacid synthase I (AHAS I) from Escherichia coli in a continuous flow reactor for production of R-phenylacetyl carbinol (R-PAC). We constructed a fusion of the large, catalytic subunit of AHAS I with a cellulose binding domain (CBD). This allowed purification of the enzyme and its immobilization on cellulose in a single step. After immobilization, AHAS I is fully active and can be used as a catalyst in an R-PAC production unit, operating either in batch or continuous mode. We propose a simplified mechanistic model that can predict the product output of the AHAS I-catalyzed reaction. This model should be useful for optimization and scaling up of a R-PAC production unit, as demonstrated by a column flow reactor.
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