Molecular annotation of ketol-acid reductoisomerases fromStreptomycesreveals a novel amino acid biosynthesis interlock mediated by enzyme promiscuity

The 6-phosphogluconate dehydrogenase superfamily oxidize and reduce a wide range of substrates, making their functional annotation challenging. Ketolacid 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

Genome mining for bioprospecting of biosynthetic genes clusters for bacterial metabolites potentially useful in agroecological production en la producción agroecológica

Objective: To describe the relevance and some tools of the genomic mining to explore genetic and molecular determinants encoded in bacterial genomes to address agronomic problems. Design/methodology/approach: Literature review of the importance of bacteria as a reservoir of biosynthetic gene clusters (BGC) involved in the production of metabolites with biological activity as anti-pathogens and of genome mining as a tool to reveal this potential. Results: The bioinformatic tools useful for the exploration of bacterial genomes has the potential to contribute to the resolution of problems in agronomy, for example, the use of bacteria, their genes and metabolites for the control of phytopathogens that attack crops of global importance. Likewise, the limitations of the genome mining and their coupling with other experimental approaches to achieve bioprospecting of BGC or their related metabolites are summarized. Limitations of the study/implications: Although the use of genome mining to explore the potential of bacteria is a very powerful approach, it will always be necessary the experimental corroboration at the laboratory level, to confirm the hypotheses generated by bioinformatics tools. Findings/conclusions: The genomic mining makes possible to take advantage of the large number of bacterial genomes currently sequenced and available in public databases to understand the genetic bases of their biological activities; as well as for the heterologous expression of biosynthetic genes, or the identification and purification of new metabolites. The foregoing with the objective of contributing with more effective and environmentally friendly solutions that address agronomic problems.Objective. To describe the relevance and some tools of genome mining to explore genetic and molecular determinants encoded in bacterial genomes to address agronomic problems. Design/Methodology/Approach. Literature review of the importance of bacteria as a reservoir of biosynthetic gene clusters (BGC), involved in the production of metabolites with biological activity as anti-pathogens; and of genome mining as a tool to reveal this potential. Results. Bioinformatic tools are useful for the exploration of bacterial genomes and have the potential to contribute to the resolution of agronomy problems. For example, the use of bacteria, their genes and metabolites for the control of phytopathogens that attack crops of global importance. Likewise, the limitations of the genome mining and their coupling with other experimental approaches to achieve bioprospecting of BGC or their related metabolites are summarized. Limitations of the study/implications: Although the use of genome mining to explore the potential of bacteria is a very powerful approach, it will always be necessary the experimental corroboration at the laboratory level, to confirm the hypotheses generated by bioinformatics tools. Findings/conclusions: Genome mining allows to take advantage of the large number of bacterial genomes currently sequenced, that are available in public databases to understand the genetic bases of their biological activities. As well as for the heterologous expression of biosynthetic genes, or the identification and purification of new metabolites. The foregoing with the objective of contributing with more effective and environmentally friendly solutions that address agronomic problems

Identification and analysis of residues contained on β → a loops of the dual-substrate (βα)8 phosphoriblosyl isomerase A specific for its phosphoribosyl anthranilate isomerase activity

A good model to experimentally explore evolutionary hypothesis related to enzyme function is the ancient-like dual-substrate (βα)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 coliin vivo complementation experiments. Structural and functional analyses of a hitherto unnoticed residue contained on the functionally important β → α loop 5, namely, Arg139, 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_Ser81Thr and PriA_Arg139Asn showed that these residues, which are contained on β → α 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_Arg139Asn elucidated at 1.95 Å herein strongly implicates the occurrence of conformational changes in this β → α 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