Mutational analysis of critical residues of FAD-independent catabolic acetolactate synthase from Enterococcus faecalis V583

Abstract

Catabolic acetolactate synthase (cALS) from Enterococcus faecalis is a FAD-independent enzyme, which catalyzes the condensation of two molecules of pyruvate to produce acetolactate. Mutational and kinetic analyses of variants suggested the importance of H111, Q112, and Q411 residues for catalysis in cALS. The wild-type and variants were expressed as equally soluble proteins and co-migrated to a size of 60 kDa on SDS-PAGE. Importantly, H111 in cALS, which is widely present as phenylalanine in many other ThDP-dependent enzymes, plays a crucial role in substrate binding. Interestingly, the H111 variants, H111R and Hill F, demonstrated altered specific activity of H111 variants with 17- and 26-fold increases in K-m, respectively, compared to wild-type cALS. Furthermore, Q112 variants, Q112E, Q112N, and Q112V, exhibited significantly lower specific activity with 70-, 15-, and 10-fold higher K-s, for ThDP, respectively. In the case of Q411, the variant Q411E showed a 10-fold rise in K-in and a 20-fold increase in K-m, for ThDP. Further, the molecular docking results indicated that the binding mode of ThDP was slightly affected in the variants of cALS. Based on these results, we suggest that H111 plays a role in substrate binding, and further suggest that Q112 and Q411 might be involved in ThDP binding of cALS. (C) 2014 Elsevier B.V. All rights reserved.