Phosphinothricin Tripeptide Synthetases in Streptomyces viridochromogenes Tü494

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Abstract
The tripeptide backbone of phosphinothricin (PT) tripeptide (PTT), a compound with herbicidal activity from Streptomyces viridochromogenes , is assembled by three stand-alone peptide synthetase modules. The enzyme PhsA (66 kDa) recruits the PT-precursor N -acetyl-demethylphosphinothricin ( N -Ac-DMPT), whereas the two alanine residues of PTT are assembled by the enzymes PhsB and PhsC (129 and 119 kDa, respectively). During or after assembly, the N-Ac-DMPT residue in the peptide is converted to PT by methylation and deacetylation. Both phsB and phsC appear to be cotranscribed together with two other genes from a single promoter and they are located at a distance of 20 kb from the gene phsA , encoding PhsA, in the PTT biosynthesis gene cluster of S. viridochromogenes . PhsB and PhsC represent single nonribosomal peptide synthetase elongation modules lacking a thioesterase domain. Gene inactivations, genetic complementations, determinations of substrate specificity of the heterologously produced proteins, and comparison of PhsC sequence with the amino terminus of the alanine-activating nonribosomal peptide synthetase PTTSII from S. viridochromogenes confirmed the role of the two genes in the bialanylation of Ac-DMPT. The lack of an integral thioesterase domain in the PTT assembly system points to product release possibly involving two type II thioesterase genes ( the1 and the2 ) located in the PTT gene cluster alone or in conjunction with an as yet unknown mechanism of product release.