Unusual transformations in the biosynthesis of the antibiotic phosphinothricin tripeptide

Abstract

Phosphinothricin-tripeptide (PTT, phosphinothricyl-alanyl-alanine) is a natural product antibiotic and potent herbicide that is produced by Streptomyces hygroscopicus ATCC 21705 1 and Streptomyces viridochromogenes DSM 40736 2 . PTT has attracted widespread interest due to its commercial applications and unique phosphinic acid functional group. Despite intensive study since its discovery in 1972 (see 3 for a comprehensive review), a number of steps early in the PTT biosynthetic pathway remain uncharacterized. Here we report a series of interdisciplinary experiments involving the construction of defined S. viridochromogenes mutants, chemical characterization of accumulated intermediates, and in vitro assay of selected enzymes to examine these critical steps in PTT biosynthesis. Our results indicate that early PTT biosynthesis involves a series of heretofore undescribed catalyses, including a highly unusual reaction for carbon bond cleavage. In sum, we define a more complex pathway for early PTT biosynthesis that includes biochemically unprecedented and chemically interesting steps. KeywordsStreptomyces viridochromogenes; phosphinothricin; biosynthesis; bialaphos; phosphonate metabolism * Corresponding Author: Department of Microbiology, University of Illinois at Urbana-Champaign, 601 South Goodwin Avenue, Urbana, IL 61801, Phone: 217-244-1943, Fax: 217-244-6697, metcalf@uiuc.edu. # Present Address: Shanghai Chemspec Corp., No. 3 Lan 1273. Accession codesSequence data from the PTT biosynthetic gene cluster that was used in designing the experiments reported here was derived solely from previously available sequence (GenBank accession number AY632421). Sequence data generated in this study that corrects apparent sequencing errors in S. hygroscopicus PTT biosynthetic genes (found in GenBank accessions AB029917.1 and D37878.1) were deposited in GenBank under accession numbers EF486265 and EF486266 respectively. The bioactive portion of the PTT (also known as bialaphos, 1) tripeptide is phosphinothricin (PT, 2). PT is the only known naturally occurring compound to incorporate a direct carbon to phosphorus to carbon (C-P-C) bond motif. PTT has become a model for the biosynthesis of phosphonic acid antibiotics based on numerous studies 3 , leading to a pathway that is regarded as mostly solved. It should be noted, however, that many of the PTT non-producing mutants analyzed to define the pathway were generated by random chemical mutagenesis and some mutations were not rigorously mapped to individual genes. Accordingly, the resulting biosynthetic model is based upon a patchwork of chemical, enzymatic, and genetic data. Further, though many PTT biosynthetic genes were previously cloned and sequenced, the full gene cluster was not completely sequenced from either producer until very recently, when the cluster from S. viridochromogenes was isolated and characterized 7,8 . Sequence analysis of the cluster revealed that only about half of the genes identified had rigorous genetic or biochemical data establishing the...

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