In vitro characterization of a heterologously expressed nonribosomal Peptide synthetase involved in phosphinothricin tripeptide biosynthesis.
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 muM(-1) min(-1), whereas the k(cat)/K(m,app) for l-N-acetyldemethylphosphinothricin was 0.5 microM(-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).
Department of Chemistry, University of Illinois, Urbana,Illinois 61801, USA.
Jin-Hee
JH
Lee
Bradley S
BS
Evans
Gongyong
G
Li
Neil L
NL
Kelleher
Wilfred A
WA
van der Donk
D000613:Aminobutyrates / Q000737:chemistry / Q000378:metabolism*; D011756:Diphosphates / Q000737:chemistry / Q000378:metabolism; D005583:Fourier Analysis; D007700:Kinetics; D013058:Mass Spectrometry; D009943:Organophosphorus Compounds / Q000737:chemistry / Q000378:metabolism; D010452:Peptide Biosynthesis; D010453:Peptide Synthases / Q000737:chemistry* / Q000378:metabolism; D013302:Streptomyces / Q000201:enzymology / Q000378:metabolism; D013379:Substrate Specificity
D000613:Aminobutyrates; D011756:Diphosphates; D009943:Organophosphorus Compounds; C046087:N-acetyldemethylphosphinothricin; C003121:phosphinothricin; D010453:Peptide Synthases; C104385:non-ribosomal peptide synthase