International Journal of Biological Macromolecules 72 (2015) 104–109 Contents lists available at ScienceDirect International Journal of Biological Macromolecules j ourna l ho me pa g e: www.elsevier.com/locate/ijbiomac Mutational analysis of critical residues of FAD-independent catabolic acetolactate synthase from Enterococcus faecalis V583 Sang-Choon Lee 1 , In-Pil Jung 1 , Irshad Ahmed Baig, Pham Ngoc Chien 2 , Im-Joung La 3 , Moon-Young Yoon ∗ Department of Chemistry, College of Natural Science, Hanyang University, Seoul 133-791, Republic of Korea a r t i c l e i n f o Article history: Received 13 March 2014 Received in revised form 2 August 2014 Accepted 4 August 2014 Available online 14 August 2014 Keywords: Enterococcus faecalis Catabolic acetolactate synthase Site-directed mutagenesis a b s t r a c t 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 H111F, 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 m and a 20-fold increase in K s 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. © 2014 Elsevier B.V. All rights reserved. 1. Introduction Acetolactate synthase (ALS) (ALS, E.C. 4.1.3.18) is a well-known thiamine diphosphate (ThDP)-dependent enzyme that catalyzes the condensation of two pyruvate molecules to produce aceto- lactate (AL), which is a key metabolic intermediate in various metabolic pathways of microorganisms [1]. Earlier studies have shown that two kinds of ALSs exist in organisms, anabolic aceto- hydroxyacid synthase (AHAS) and catabolic ALSs (cALS) [2]. 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) as a cofactor [3]. However, the FAD have only structural role in AHAS, which is required solely for maintaining the enzyme active site in the required geometry for catalysis to occur. The AHAS has been ∗ Corresponding author. Tel.: +82 2 2220 0946; fax: +82 2 2299 0763. E-mail address: myyoon@hanyang.ac.kr (M.-Y. Yoon). 1 These authors contributed equally to this work. 2 Present address: Department of Bioengineering, Hanyang University, Seoul 133- 791, Republic of Korea. 3 Present address: Food Safety Center, Lotte Confectionery Co., Ltd., Seoul 150-866, Republic of Korea. remained an attractive target for many commercial herbicides and developing antimicrobials [25–29]. In contrast to AHAS, the cALS is found only in some bacteria and is involved in the butanediol fermentation pathway [4,5]. Both enzymes essentially require ThDP and the divalent metal ion Mg 2+ to exhibit catalytic activity. Like in AHAS or other ThDP-dependent enzymes, ThDP is located centrally in the active site of cALS with a unique V-conformation at the dimer interface to play a central role of intramolecular proton transfer in the catalytic cycle. In catalysis, a divalent metal ion Mg 2+ serves to anchor the diphosphate moiety of ThDP at the active site of cALS. However, despite these similarities, cALS has a lower pH optimum of about 6.0, has FAD-independent functionality, its genetic location is within the butanediol operon, and it lacks a regulatory subunit compared to AHAS [6–8]. The cALS from some bacterial sources have been purified and characterized previously [9–12]. The only reported crystal structure of cALS, from Klebsiella pneumoniae [13], reveals the location and conformation of the active site and the binding site of cofactors ThDP and Mg 2+ . It is noteworthy that, despite the overall similarity in the crystal structure of cALS and AHAS, the essential cofactor ThDP, which has a unique tricyclic conformation in cALS, has never been observed in many of the reported three-dimensional structures of ThDP-dependent enzymes [13]. Furthermore, the crystal structure of cALS from K. pneumoniae revealed significant structural changes http://dx.doi.org/10.1016/j.ijbiomac.2014.08.002 0141-8130/© 2014 Elsevier B.V. All rights reserved.