Molecular annotation of ketol‐acid reductoisomerases from S treptomyces reveals a novel amino acid biosynthesis interlock mediated by enzyme promiscuity

Summary The 6‐phosphogluconate dehydrogenase superfamily oxidize and reduce a wide range of substrates, making their functional annotation challenging. Ketol‐acid reductoisomerase ( KARI ), encoded by the ilvC gene in branched‐chain amino acids biosynthesis, is a promiscuous reductase enzyme within this superfamily. Here, we obtain steady‐state enzyme kinetic parameters for 10 IlvC homologues from the genera S treptomyces and C orynebacterium , upon eight selected chemically diverse substrates, including some not normally recognized by enzymes of this superfamily. This biochemical data suggested a S treptomyces biosynthetic interlock between proline and the branched‐chain amino acids, mediated by enzyme substrate promiscuity, which was confirmed via mutagenesis and complementation analyses of the proC , ilvC 1 and ilvC 2 genes in S treptomyces coelicolor . Moreover, both ilvC orthologues and paralogues were analysed, such that the relationship between gene duplication and functional diversification could be explored. The KARI paralogues present in S . coelicolor and S treptomyces lividans , despite their conserved high sequence identity (97%), were shown to be more promiscuous, suggesting a recent functional diversification. In contrast, the KARI paralogue from S treptomyces viridifaciens showed selectivity towards the synthesis of valine precursors, explaining its recruitment within the biosynthetic gene cluster of valanimycin . These results allowed us to assess substrate promiscuity indices as a tool to annotate new molecular functions with metabolic implications.