| Phosphinothricin-tripeptide (PTT) is a non-ribosomally produced peptide antibiotic and herbicide produced by Streptomyces viridochromogenes and Streptomyces hygroscopicus. Research interest in PTT stems from its commercially exploited herbicidal activity and from its unique C-P-C bond motif. PTT biosynthesis has largely been elucidated in the decades subsequent to its discovery in the early 1970s, but multiple steps early in the biosynthetic pathway had not been adequately investigated despite its use as a model pathway for reduced-phosphorus antibiotic biosynthesis.; To define and characterize the early steps in PTT biosynthesis that comprise the stepwise conversion of phosphonoacetaldehyde to carboxyphosphonoenolpyruvate, the PTT gene cluster was first isolated and sequenced after it was heterologously expressed in Streptomyces lividans. This was done because the intact gene cluster had not previously been isolated and sequenced from either producing organism. New PTT biosynthetic genes were discovered within the cluster and the functions of these genes and their encoded proteins were elucidated by combined biochemical and genetic analyses using techniques in multiple bacterial hosts.; Our investigations led to the discovery of new biosynthetic intermediates and revealed inaccuracies in the previously published pathway. Specifically, this thesis work established that step III of PTT biosynthesis is the reduction of phosphonoacetaldehyde to hydroxyethylphosphonate by PhpC, an alcohol dehydrogenase with a previously unrecognized role in PTT biosynthesis. We also demonstrated that step IV of PTT biosynthesis is the conversion of hydroxyethylphosphonate to hydroxymethylphosphonate in a chemically novel reaction catalyzed by PhpD, a protein with no recognizable homologs in GenBank. We found evidence to suggest that step V of PTT biosynthesis is the oxidation of hydroxymethylphosphonic acid to phosphonoformaldehyde by PhpE, an alcohol dehydrogenase. Phosphonoformaldehyde is likely then converted into the established intermediate phosphonoformate by the aldehyde dehydrogenase PhpJ (step VI). We demonstrated that phosphonoformate is activated by conjugation to CTP in a reaction catalyzed by PhpF that produces the previously undescribed intermediate CMP-5'-phosphonoformate. Finally, we presented evidence to suggest that CMP-5'-phosphonoformate is likely required for the net incorporation of phosphonoformate into carboxyphosphonoenolpyruvate. This body of work has important implications in our understanding of phosphonic acid antibiotic biosynthesis, a group with great therapeutic promise. |
