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42064-G4
Carbon-Phosphorus Bond-Containing Natural Peptides
Carbon-phosphorus (C–P) bond-containing natural products are remarkable for their diverse and potent biological activities and unique roles in primary and secondary metabolism. Examples of clinically important enzymatic targets of natural phosphonates and related compounds include glutamine synthetase, beta-lactamase, angiotensin converting enzyme (ACE) and mevalonic acid biosynthesis which result in herbicidal, antibacterial, antihypertensive and antiparasitic activity, respectively. These activities frequently derive from the ability of phosphonates to resemble enzymatic reaction transition states and to compete with biological phosphates, which often play essential roles in substrate binding and enzyme catalysis.
Increasing numbers of naturally occurring phosphonates have been identified in recent years. The biosynthetic origins of several C P bond containing natural products have been investigated and in all cases thus far examined, the C–P bond has been formed by rearrangement of phosphoenolpyruvate to phosphonopyruvate or an analogous process. Of interest to us is the tripeptide K-26 from an actinomycete, representative of an uninvestigated class of natural phosphonates which incorporate a phosphonic acid analog of tyrosine, (R)-1-amino-2-(4-hydroxyphenyl)ethylphosphonic acid (AHEP).4 K-26 is one of the most potent natural product ACE inhibitor reported, with an IC50 value of 12 nM and good in vivo efficacy.
One overall all aim of this project is to determine the true mechanism of C P bond formation, be it via tyrosine, a closely related metabolite or a tripeptide precursor. Due to the unprecedented structure of K-26 we have engaged in a multifaceted approach for the elucidation of K-26 biosynthesis. Classical isotope incorporation studies (using new methodologies developed by us) have revealed primary metabolic precursors and secondary metabolic biosynthetic intermediates. AHEP is a discrete intermediate in the biosynthesis of K-26 and tyrosine is a close precursor of AHEP. Tyrosine is not deaminated but it is decarboxylated en route to AHEP, but not via tyramine. The intermediacy of AHEP suggests that this nonproteinogenic amino acid is appended to a peptide precursor via a nonribosomal mechanism. These data unambiguously establish that the C-P bond in K-26 is biosynthesized by a unprecedented (non phosphoenolpyruvate based). When fully described, the biosynthetic machinery will contain at least one new class of bond forming enzyme.
Genetic investigations, guided by these precursor identification studies, have revealed the genetic 'blueprints' for K-26 biosynthesis. We have obtained a high resolution sequence of the genome and a 10,000 member fosmid library covering the entire genome. Over the past year we have annotated the majority of the secondary metabolic genes identified by us in the producing stain. As a result of this detailed investigation, we have identified two non-ribosomal peptide synthetase (NRPS) gene clusters that we consider as candidates for K-26 biosynthesis. Each gene cluster is a trimodular NRPS encoding the biosynthesis of a tripeptide. The sequence signatures of the NRPS are partially consistent with the expected signatures for K-26 biosynthesis.
