Characterization of two 2-isopropylmalate synthase homologs from Thermus thermophilus HB27 Ayako Yoshida a , Saori Kosono a, b, c , Makoto Nishiyama a, b, * a Biotechnology Research Center, The University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan b Collaborative Research Institute for Innovative Microbiology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan c RIKEN Center for Sustainable Resource Science, 2-1, Hirosawa, Wako-shi, Saitama, 351-0198, Japan article info Article history: Received 18 April 2018 Accepted 2 May 2018 Available online xxx Keywords: 2-Isopropylmalate synthase Citramalate synthase Thermus thermophilus BCAA biosynthesis abstract 2-Isopropylmalate synthase (IPMS) catalyzes the first step of leucine biosynthesis and is regulated via feedback inhibition by leucine. The thermophilic bacterium, Thermus thermophilus HB27, has two IPMS homologous genes: TTC0847 and TTC0849, both of which are in the branched-chain amino acid biosynthetic gene cluster. Since enzymes involved in the leucine biosynthetic pathway are evolutionarily related to those in isoleucine biosynthesis, TTC0847 and TTC0849 are expected to function as IPMS or citramalate synthase, which is the first enzyme in the isoleucine biosynthetic pathway from pyruvate. We characterized these proteins in vitro and in vivo, and revealed that TTC0849 plays a key role in the biosynthesis of leucine and isoleucine, whereas TTC0847 is only involved in that of isoleucine. © 2018 Elsevier Inc. All rights reserved. 1. Introduction In bacteria, 2-isopropylmalate synthase (IPMS) is a key enzyme in leucine biosynthesis, catalyzing the formation of 2- isopropylmalate from 2-oxoisovalerate and acetyl-CoA by Claisen- like condensation [1 ,2]. IPMS belongs to the protein family called the DRE_TIM_metallolyase superfamily with a TIM barrel structure, which includes homocitrate synthase in lysine biosynthesis using 2-oxoglutarate as a substrate [3], citramalate synthase (CMS) in isoleucine biosynthesis using pyruvate [4], re-citrate synthase in the TCA cycle [5], and methylthioalkylmalate synthase in glucosi- nolate biosynthesis [6]. These enzymes catalyze the metal- dependent addition of an acetyl group of acetyl-CoA to the carbonyl moiety of a-ketoacid substrates and are considered to have evolved from the same ancestral enzyme. 2-Isopropylmalate synthesized by IPMS is converted to leucine by the following en- zymes: 3-isopropylmalate dehydratase (LeuCD), 3-isopropylmalate dehydrogenase (IPMDH), and branched-chain amino acid (BCAA) aminotransferase [7](Fig. 1A). Two biosynthetic pathways for isoleucine have been identified to date; one is the pyruvate pathway that utilizes pyruvate as a starting material using CMS as the first enzyme, and the other pathway uses threonine as a starter, which is converted to 2- oxobutanoate by threonine dehydratase. The pyruvate pathway was initially suggested in the methanogenic archaea, Meth- anococcus jannaschii [4], and has been reported in several organ- isms [8,9]. IPMS and CMS catalyze a similar reaction and share a similar domain structure with an N-terminal TIM barrel domain and C-terminal LeuA-dimer. In the amino acid biosynthetic pathway, IPMS and CMS are generally inhibited by leucine and isoleucine, respectively, in a feedback manner [10]. Crystal struc- ture studies revealed that the C-terminal LeuA-dimer binds the cognate inhibitor and functions as a regulatory domain [11 , 12]. Furthermore, mutational studies proposed that subdomain II in the linker domain connecting the N-terminal catalytic domain with a TIM barrel structure and C-terminal regulatory domain called the LeuA-dimer was important for the catalytic and regulatory func- tions of IPMS [13e15]. In the present study, we characterized the functions of two IPMS homologs TTC0847 and TTC0849, which are located in the BCAA biosynthetic gene cluster (TTC0847- TT0852) in Thermus thermo- philus HB27 (Fig. 1B). TTC0847 and TTC0849 share 33% amino acid identity and have a typical IPMS domain organization with an N- terminal catalytic domain and C-terminal regulatory domain. Genomic information indicates that one of these genes functions as IPMS and the other as CMS (Fig. 1A). In the present study, we revealed that TTC0849 exhibited dual activities as IPMS and CMS, whereas TTC0847 only showed CMS activity. Our results also * Corresponding author. Biotechnology Research Center, The University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan. E-mail address: umanis@mail.ecc.u-tokyo.ac.jp (M. Nishiyama). Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc https://doi.org/10.1016/j.bbrc.2018.05.013 0006-291X/© 2018 Elsevier Inc. All rights reserved. Biochemical and Biophysical Research Communications xxx (2018) 1e6 Please cite this article in press as: A. Yoshida, et al., Characterization of two 2-isopropylmalate synthase homologs from Thermus thermophilus HB27, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.05.013