In Vitro Characterization of a Heterologously Expressed Nonribosomal Peptide Synthetase Involved in Phosphinothricin Tripeptide Biosynthesis

[Image: see text] The late stages of biosynthesis of phosphinothricin tripeptide (PTT) involve peptide formation and methylation on phosphorus. The exact timing of these transformations is not known. To provide insight into this question, we developed a heterologous expression system for PhsA, one of three NRPS proteins in PTT biosynthesis. The apparent k(cat)/K(m) value for ATP−pyrophosphate exchange activity for d,l-N-acetylphosphinothricin was 3.5 μM(−1) min(−1), whereas the k(cat)/K(m,app) for l-N-acetyldemethylphosphinothricin was 0.5 μM(−1) min(−1), suggesting the former might be the physiological substrate. Each substrate could be loaded onto the phosphopantetheine arm of the thiolation domain as observed by Fourier transform mass spectrometry (FTMS).