2-Isopropylmalate synthase (IPMS) catalyzes the first step of leucine biosynthesis and is regulated via feedback inhibition by leucine. The thermophilic bacterium, Thermus thermophilus H827, 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. (C) 2018 Elsevier Inc. All rights reserved.