Role of a Highly Conserved and Catalytically Important Glutamate-49 in the Enterococcus faecalis Acetolactate Synthase

Abstract

Acetolactate synthase (ALS) is a thiamine diphosphate (ThDP)-dependent enzyme that catalyzes the decarboxylation of pyruvate and then condenses the hydroxyethyl moiety with another molecule of pyruvate to give 2-acetolactate (AL). AL is a key metabolic intermediate in various metabolic pathways of microorganisms. In addition, AL can be converted to acetoin, an important physiological metabolite that is excreted by many microorganisms. There are two types of ALSs reported in the literature, anabolic acetohydroxyacid synthase (AHAS) and catabolic ALSs (cALS). The anabolic AHAS is primarily found in plants, fungi, and bacteria, is involved in the biosynthesis of branched-chain amino acids (BCAAs), and contains flavin adenine dinucleotide (FAD), whereas the cALS is found only in some bacteria and is involved in the butanediol fermentation pathway. Both of the enzymes are ThDP-dependent and require a divalent metal ion for catalytic activity. Despite the similarities of the reactions catalyzed, the cALS can be distinguished from anabolic AHAS by a low optimal pH of about 6.0, FAD-independent functionality, a genetic location within the butanediol operon, and lack of a regulatory subunit. It is noteworthy that the structural and functional features of AHAS have been extensively studied, in contrast to those of cALS, for which only limited information is available. To date, the only crystal structure of cALS reported is from Klebsiella pneumonia, which revealed that the overall structure of K. pneumonia ALS is similar to that of AHAS except for the FAD binding region found in AHAS. The recent discovery of the crystal structures of various ThDP-dependent enzymes, including cALS, has greatly aided the understanding of the overall structural orientation of ThDP and its role in catalysis. As with the other ThDPdependent enzymes, ThDP is located in the active site of cALS with a unique V-conformation at the subunit-subunit interface, where the diphosphate moiety of ThDP is in contact with one subunit and the aminopyrimidine moiety is in contact with the other subunit. In all of the crystal structures of ThDP-dependent enzymes determined to date, with the exception of glyoxylate carbo-ligase (GCL), a glutamate residue has been found at hydrogen-bonding distance from the N1' atom of the aminopyrimidine ring of the bound ThDP and plays a key role in catalysis (Fig. 1). In addition to ThDP binding, the glutamate interaction with the N1' atom of the ThDP aminopyrimidine moiety is involved in activation of the cofactor for proton exchange in several ThDP-dependent enzymes and induces the formation ofe 1',4'-iminotautomer, which generates the highly reactive ylide required for catalysis. This glutamate residue was found to be highly conserved in almost all of the ThDP-dependent family of enzymes (Fig. 2). Subsequently, the crucial catalytic role of this glutamate residue was extensively studied by site-directed mutagenesis in a few ThDP-dependent enzymes, including AHAS and pyruvate decarboxylase (PDC). Clearly, it is also very important to investigate the extent to which this glutamate is essential in other ThDP-dependent enzymes. In this study we investigated the influence of this catalytically important glutamate residue (Glu-49) on both catalysis and cofactor binding in cALS of Enterococcus faecalis. We recently cloned, purified, and characterized the cALS of E. faecalis. Site-directed mutagenesis was carried