Streptomyces lividans 66 produces a protease inhibitor via a tRNA-utilizing enzyme interacting with a C-minus NRPS.

Small peptide aldehydes (SPAs) with protease inhibitory activity are naturally occurring compounds shown to be synthesized by non-ribosomal peptide synthetases (NRPS). SPAs are widely used in biotechnology and have been utilized as therapeutic agents. They are also physiologically relevant and have been postulated to regulate the development of their producing microorganisms. Previously, we identified an NRPS-like biosynthetic gene cluster (BGC) in Streptomyces lividans 66 that lacked a condensation (C) domain but included a tRNA-utilizing enzyme (tRUE) belonging to the leucyl/phenylalanyl (L/F) transferase family. This system was predicted to direct the synthesis of a novel SPA, which we named livipeptin. Using evolutionary genome mining approaches, here, we confirm the presence of L/F transferase tRUEs within the genomes of diverse Streptomyces and related organisms, including fusions with the anticipated C-minus NRPS-like protein. We then demonstrate genetic functional cooperation between the identified L/F-transferase divergent tRUE homolog with the C-minus NRPS, leading to the synthesis of a metabolic fraction with protease inhibitory activity. Semisynthetic assays in the presence of RNAse revealed that the productive interaction between the tRUE and the C-minus NRPS enzymes is indeed tRNA dependent. We expect our findings to boost the discovery of SPAs, as well as the development of protease-mediated biotechnologies, by exploiting the uncovered genetic basis for synthesizing putative acetyl-leu/phe-arginine protease inhibitors. Furthermore, these results will facilitate the purification and structural elucidation of livipeptin, which has proven difficult to chemically characterize. SIGNIFICANCE: The discovery of natural products biosynthetic genes marks a significant advancement in our understanding of these metabolites, for example of their evolution, activity, and biosynthesis, but also opens biotechnological opportunities and knowledge to advance genome mining approaches. We made this possible by uncovering a new biosynthetic pathway in Streptomyces lividans 66 shown to direct the synthesis of a strong protease inhibitor, termed livipeptin, following unprecedented biosynthetic rules and genes. Thus, by shedding light on the genetic mechanisms predicted to govern the production of acetyl-leu/phe-arginine protease inhibitors, including the elusive livipeptin, this study enables novel protease-mediated biotechnologies as well as approaches for discovering protease inhibitors from genome data.

Molecular annotation of ketol-acid reductoisomerases from Streptomyces reveals 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. 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.

Exploring the Structure-Function Loop Adaptability of a (ß/α)(8)-Barrel Enzyme through Loop Swapping and Hinge Variability.

Evolution of proteins involves sequence changes that are frequently localized at loop regions, revealing their important role in natural evolution. However, the development of strategies to understand and imitate such events constitutes a challenge to design novel enzymes in the laboratory. In this study, we show how to adapt loop swapping as semiautonomous units of functional groups in an enzyme with the (ß/α)(8)-barrel and how this functional adaptation can be measured in vivo. To mimic the natural mechanism providing loop variability in antibodies, we developed an overlap PCR strategy. This includes introduction of sequence diversity at two hinge residues, which connect the new loops with the rest of the protein scaffold, and we demonstrate that this is necessary for a successful exploration of functional sequence space. This design allowed us to explore the sequence requirements to functional adaptation of each loop replacement that may not be sampled otherwise. Libraries generated following this strategy were evaluated in terms of their folding competence and their functional proficiency, an observation that was formalized as a Structure-Function Loop Adaptability value. Molecular details about the function and structure of some variants were obtained by enzyme kinetics and circular dichroism. This strategy yields functional variants that retain the original activity at higher frequencies, suggesting a new strategy for protein engineering that incorporates a more divergent sequence exploration beyond that limited to point mutations. We discuss how this approach may provide insights into the mechanism of enzyme evolution and function.

Identification and analysis of residues contained on beta --> alpha loops of the dual-substrate (beta alpha)8 phosphoribosyl 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 (beta alpha)(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 beta --> alpha 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 beta --> alpha 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 A herein strongly implicates the occurrence of conformational changes in this beta --> alpha 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.