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A good model to experimentally explore evolutionary hypothesis related to enzyme function is the ancient-like dual-substrate (ba) 8 phosphoribosyl isomerase A (PriA), which takes part in both histidine and tryptophan biosynthesis in Streptomyces coelicolor and related organisms. In this study, we determined the Michaelis-Menten enzyme kinetics for both isomerase activities in wild-type PriA from S. coelicolor and in selected single-residue monofunctional mutants, identified after Escherichia coli in vivo complementation experiments. Structural and functional analyses of a hitherto unnoticed residue contained on the functionally important b fi a loop 5, namely, Arg 139 , which was postulated on structural grounds to be important for the dual-substrate specificity of PriA, is presented for the first time. Indeed, enzyme kinetics analyses done on the mutant variants PriA_Ser 81 Thr and PriA_Arg 139 Asn showed that these residues, which are contained on b fi a loops and in close proximity to the N-terminal phosphate-binding site, are essential solely for the phosphoribosyl anthranilate isomerase activity of PriA. Moreover, analysis of the X-ray crystallographic structure of PriA_Arg 139 Asn elucidated at 1.95 Å herein strongly implicates the occurrence of conformational changes in this b fi a loop as a major structural feature related to the evolution of the dual-substrate specificity of PriA. It is suggested that PriA has evolved by tuning a fine energetic balance that allows the sufficient degree of structural flexibility needed for accommodating two topologically dissimilar substrates-within a bifunctional and thus highly constrained active site-without compromising its structural stability.

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.

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Conocimiento previo/especie: Agave tequilana Weber var. azul es un importante cultivo en México, utilizado para la producción de tequila. Muchas especies de Agave son tolerantes a condiciones áridas. Sin embargo, las bases moleculares de los mecanismos seleccionados en los agaves para confrontar el estrés abiótico, no han sido descritas. Hipótesis: Las proteínas abundantes en la embriogénesis tardía (LEAPs), una superfamilia asociada a las respuestas ante el estrés abiótico en plantas, son un elemento clave en las respuestas de los agaves ante ambientes áridos. Métodos: Datos transcriptómicos de A. tequilana fueron utilizados para realizar análisis in silico e identificar genes que codifican Agave LEAPs. Comparamos sus características estructurales y su similitud/divergencia con LEAPs de otras plantas, utilizando bioinformática. La abundancia de los transcritos de AteqLEAP en órganos vegetativos y en respuesta a altas temperaturas fue determinada mediante qRT-PCR. Resultados: Identificamos tres AteqLEAPs estructuralmente diferentes. Las AteqLEA_5Bs muestran similitud (relativamente baja) con LEAPs conocidas como “atípicas” (LEA_3) y exhiben, inesperadamente, altos niveles de expresión constitutiva en hojas. Los transcritos de AteqLEA_5C (LEA_2) mostraron baja expresión en todos los órganos analizados. Dos isoformas de AteqDHN tipo SK3 muestran el típico desorden estructural e hidrofilicidad de las dehidrinas y son altamente expresadas en hojas no desarrolladas, meristemo vegetativo y tallo (piña). Conclusiones: Las AteqLEAP_5B parecen tener un papel protector preventivo en las hojas fotosintéticas plenamente funcionales; mientras que las AteqDHNs parecen proteger tejidos en proceso de diferenciación como meristemos y hojas en desarrollo; así como tejidos de almacenamiento, como el tallo del agave.