title: Molecular annotation of ketol-acid reductoisomerases from Streptomyces reveals a novel amino acid biosynthesis interlock mediated by enzyme promiscuity.
abstract: 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 Streptomyces and Corynebacterium, upon eight selected chemically diverse substrates, including some not normally recognized by enzymes of this superfamily. This biochemical data suggested a Streptomyces 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, ilvC1 and ilvC2 genes in Streptomyces 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 Streptomyces 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 Streptomyces 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.
authors.affiliation: Evolution of Metabolic Diversity Laboratory, Unidad de Genómica Avanzada (Langebio), Cinvestav-IPN, Km 9.6 Libramiento Norte, Irapuato, Guanajuato, CP36822, México.
authors.forename: Karina
authors.initials: K
authors.lastname: Verdel-Aranda
authors.forename: Susana T
authors.initials: ST
authors.lastname: López-Cortina
authors.forename: David A
authors.initials: DA
authors.lastname: Hodgson
authors.forename: Francisco
authors.initials: F
authors.lastname: Barona-Gómez
mesh_terms: D000596:Amino Acids / Q000096:biosynthesis*; D053898:Biosynthetic Pathways / Q000235:genetics*; D003352:Corynebacterium / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D055786:Gene Knockout Techniques; D005816:Genetic Complementation Test; D014644:Genetic Variation; D050540:Ketol-Acid Reductoisomerase / Q000235:genetics* / Q000378:metabolism*; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D000596:Amino Acids; D050540:Ketol-Acid Reductoisomerase