A Specialized Dehydrogenase Provides l‐Phenyllactate for FR900359 Biosynthesis

d‐Phenyllactate (PLA) is a component of the selective Gq protein inhibitor and nonribosomal cyclic depsipeptide FR900359 (FR). Here we report a detailed biochemical investigation of PLA biosynthesis and its incorporation into the natural product FR. The enzyme FrsC, member of the lactate/malate dehydrogenase superfamily, was shown to catalyze the formation of l‐PLA from phenylpyruvate. FrsC was kinetically characterized and its substrate specificity determined. Incorporation of l‐PLA was probed by assaying the adenylation domain FrsE‐A3 and feeding studies with a Chromobacterium vaccinii ΔfrsC mutant, confirming preferred activation of l‐PLA followed by on‐line epimerization to d‐PLA. Finally, detailed bioinformatic analyses of FrsC revealed its close relation to malate dehydrogenases from primary metabolism and suggest extensions in the substrate binding loop to be responsible for its adaptation to accepting larger aromatic substrates with high specificity. The dehydrogenase FrsC is involved in biosynthesis of the Gq protein inhibiting natural product FR900359. Here, we biochemically characterized FrsC, catalyzing the formation of l‐phenyllactate, and its subsequent incorporation into the FR assembly line followed by epimerization. A detailed bioinformatic analysis revealed that FrsC has evolved from primary metabolic enzymes to a specialized reductase accepting only aromatic substrates.