Although natural products have been a particularly rich source of human medicines, activity-based screening results in a very high rate of rediscovery of known molecules. Based on the large number of natural product biosynthetic genes in microbial genomes, many have proposed "genome mining" as an alternative approach for discovery efforts; however, this idea has yet to be performed experimentally on a large scale. Here, we demonstrate the feasibility of large-scale, high-throughput genome mining by screening a collection of over 10,000 actinomycetes for the genetic potential to make phosphonic acids, a class of natural products with diverse and useful bioactivities. Genome sequencing identified a diverse collection of phosphonate biosynthetic gene clusters within 278 strains. These clusters were classified into 64 distinct groups, of which 55 are likely to direct the synthesis of unknown compounds. Characterization of strains within five of these groups resulted in the discovery of a new archetypical pathway for phosphonate biosynthesis, the first (to our knowledge) dedicated pathway for H-phosphinates, and 11 previously undescribed phosphonic acid natural products. Among these compounds are argolaphos, a broad-spectrum antibacterial phosphonopeptide composed of aminomethylphosphonate in peptide linkage to a rare amino acid N 5 -hydroxyarginine; valinophos, an N-acetyl L-Val ester of 2,3-dihydroxypropylphosphonate; and phosphonocystoximate, an unusual thiohydroximate-containing molecule representing a new chemotype of sulfur-containing phosphonate natural products. Analysis of the genome sequences from the remaining strains suggests that the majority of the phosphonate biosynthetic repertoire of Actinobacteria has been captured at the gene level. This dereplicated strain collection now provides a reservoir of numerous, as yet undiscovered, phosphonate natural products.natural products | genome mining | phosphonic acid | antibiotic
Streptomyces regensis strain WC-3744 was identified as a potential phosphonic acid producer in a large-scale screen of microorganisms for the presence of the pepM gene, which encodes the key phosphonate biosynthetic enzyme phosphoenolpyruvate phosphonomutase. 31P NMR revealed the presence of several unidentified phosphonates in spent medium after growth of S. regensis. These compounds were purified and structurally characterized via extensive 1D and 2D NMR spectroscopic and mass spectrometric analyses. Three new phosphonic acid metabolites, whose structures were confirmed by comparison to chemically synthesized standards, were observed: (2-acetamidoethyl)phosphonic acid (1), (2-acetamido-1-hydroxyethyl)phosphonic (3), and a novel cyanohydrin-containing phosphonate, (cyano(hydroxy)methyl)phosphonic acid (4). The gene cluster responsible for synthesis of these molecules was also identified from the draft genome sequence of S. regensis, laying the groundwork for future investigations into the metabolic pathway leading to this unusual natural product.
Tyrosine recombinases mediate a wide range of important genetic rearrangement reactions. Models for tyrosine recombinases have been based largely on work done on the integrase of phage lambda and recombinases like Cre, Flp, and XerC/D. All of these recombinases share a common amino acid signature that is important for catalysis. Several conjugative transposons (CTns) encode recombinases that are also members of the tyrosine recombinase family, but the reaction that they catalyze differs in that recombination does not require homology in the attachment sites. In this study, we examine the role of the core-binding (CB) domain of the CTnDOT integrase (IntDOT) that is located adjacent to the catalytic domain of the protein. Since there is no crystal structure for any of the CTn integrases, we began with a predicted three-dimensional structure produced by homology-based modeling. Amino acid substitutions were made at positions predicted by the model to be close to the DNA. Mutant proteins were tested for the ability to mediate integration in vivo and for in vitro DNA-binding, cleavage, and ligation activities. We identified for the first time nonconserved amino acid residues in the CB domain that are important for catalytic activity. Mutant proteins with substitutions at three positions in the CB domain are defective for DNA cleavage but still proficient in ligation. The positions of the residues in the complex suggest that the mutant residues affect the positioning of the cleaved phosphodiester bond in the active site without disruption of the ligation step.Conjugative transposons (CTns), also known as integrative and conjugative elements, are DNA elements that can excise from the bacterial chromosome as a double-stranded circle and transfer by conjugation to another bacterial host, where they integrate into the recipient chromosome. CTnDOT is an example of a CTn that resides in the Bacteroides chromosome. The integrase of CTnDOT (IntDOT), which is essential for both excision and integration, was classified as a member of the tyrosine recombinase family (17) based on identification of five of the six active site amino acids that are conserved in tyrosine recombinases, RK(H/K)R(H/W)Y, which perform the chemistry involved in the cleavage and ligation during CTnDOT integration. One of the essential amino acids was the predicted catalytic tyrosine. Previously, we demonstrated that changing three of the six conserved amino acids eliminated integration of IntDOT in vivo and abolished or reduced cleavage and ligation activity in vitro (17). In other tyrosine recombinases, changes in all six of these conserved amino acids eliminated or drastically reduced recombination (for a review, see reference 29). Our mutational analysis of the catalytic region of IntDOT suggested that the architecture of the active site is similar to that of other tyrosine recombinases, such as the integrase of phage ( Int), Cre, Flp, and XerC/D. However, IntDOTcatalyzed recombination is significantly different because it is not site specific and tole...
Several oxime-containing small molecules have useful properties, including antimicrobial, insecticidal, anticancer, and immunosuppressive activities. Phosphonocystoximate and its hydroxylated congener, hydroxyphosphonocystoximate, are recently discovered oxime-containing natural products produced by sp. NRRL S-481 and NRRL WC-3744, respectively. The biosynthetic pathways for these two compounds are proposed to diverge at an early step in which 2-aminoethylphosphonate (2AEPn) is converted to ()-1-hydroxy-2-aminoethylphosphonate (()-1H2AEPn) in but not in sp. NRRL S-481). Subsequent installation of the oxime moiety into either 2AEPn or ()-1H2AEPn is predicted to be catalyzed by PcxL or HpxL from sp. NRRL S-481 and NRRL WC-3744, respectively, whose sequence and predicted structural characteristics suggest they are unusual -oxidases. Here, we show that recombinant PcxL and HpxL catalyze the FAD- and NADPH-dependent oxidation of 2AEPn and 1H2AEPn, producing a mixture of the respective aldoximes and nitrosylated phosphonic acid products. Measurements of catalytic efficiency indicated that PcxL has almost an equal preference for 2AEPn and ()-1H2AEPn. 2AEPn was turned over at a 10-fold higher rate than ()-1H2AEPn under saturating conditions, resulting in a similar but slightly lower / We observed that ()-1H2AEPn is a relatively poor substrate for PcxL but is clearly the preferred substrate for HpxL, consistent with the proposed biosynthetic pathway in HpxL also used both 2AEPn and ()-1H2AEPn, with the latter inhibiting HpxL at high concentrations. Bioinformatic analysis indicated that PcxL and HpxL are members of a new class of oxime-forming -oxidases that are broadly dispersed among bacteria.
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