Synthesis of 4-methylvaleric acid, a precursor of pogostone, involves a 2-isobutylmalate synthase related to 2-isopropylmalate synthase of leucine biosynthesis

SUMMARY We show here that the side chain of pogostone, one of the major components of patchouli oil obtained from Pogostemon cablin and possessing a variety of pharmacological activities, is derived from 4-methylvaleric acid. We also show that 4-methylvaleric acid is produced through the one-carbon α- ketoacid elongation pathway with the involvement of the key enzyme 2- isobutylmalate synthase (IBMS), a newly identified enzyme related to isopropylmalate synthase (IPMS) of Leu biosynthesis. Site-directed mutagenesis identified Met 132 in the N-terminal catalytic region as affecting the substrate specificity of PcIBMS1. And even though PcIBMS1 possesses the C-terminal domain that in IPMS serves to mediate Leu inhibition, it is insensitive to Leu. The observation of the evolution of IBMS from IPMS, as well as previously reported examples of IPMS-related genes involved in making glucosinolates in Brassicaceae, acylsugars in Solanaceae, and flavor compounds in apple, indicate that IPMS genes represent an important pool for the independent evolution of genes for specialized metabolism. One Sentence Summary We describe a novel enzyme, 2-isobutylmalate synthase, that is related to 2-isopropylmalate synthase and is able to efficiently convert 4-methyl-2- oxovalerate to 2-isobutylmalate, a key intermediate in the synthesis of the pogostone precursor 4-methylvaleric acid.