Characterizing Proteins for the First Committed Step in a New Biosynthetic Pathway for Ester Synthesis in Ripening Apple Fruit

In ripening apple fruit, we propose a new pathway that uses the starting products pyruvate and acetyl-CoA for the synthesis of precursors to branched- and straight-chain esters. This pathway not only provides for the synthesis of isoleucine, but also for 3-, 4-, and 5- carbon fatty acids via the process of single-carbon fatty acid elongation. Our previous apple disc feeding studies, which documented the incorporation of ¹³C- labeled acetate into isoleucine and esters containing 2-methylbutanoate, a degradation product of isoleucine, supports this hypothesis.  We identified two novel genes from apple, the sequence of which suggests that they are members of the 2-isopropylmalate synthase (IPMS) gene family.  Purified His-tag protein from these genes was found to form citramalate and 2-ethylmalate from the α-keto acids pyruvate and α-ketobutyrate, respectively, when acetyl-CoA was added.  Substrate specificity for α-keto acids in decreasing order was α-ketobutyrate, pyruvate, α-ketovalerate, and α-ketoisovalerate.  IPMS differs from this protein in that it has a much lower preference for pyruvate. We therefore hypothesize the genes (MdCIM1 and MdCIM2) are alleles coding for citramalate synthase, which contributes to carbon flux via a new pathway for ester biosynthesis.  The hypothesized pathway constitutes a conceptual shift in the regulation of ester biosynthesis in that it implies synthetic, rather than catabolic pathways are responsible for ester precursor supply.