In vitro characterization of a heterologously expressed nonribosomal peptide synthetase involved in phosphinothricin tripeptide biosynthesis

Jin Hee Lee; Bradley S. Evans; Gongyong Li; Neil L. Kelleher; Wilfred A. Van Der Donk

doi:10.1021/bi900164d

In vitro characterization of a heterologously expressed nonribosomal peptide synthetase involved in phosphinothricin tripeptide biosynthesis

Jin Hee Lee, Bradley S. Evans, Gongyong Li, Neil L. Kelleher, Wilfred A. Van Der Donk

Molecular Biosciences

Research output: Contribution to journal › Article › peer-review

15 Scopus citations

Abstract

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).

Original language	English (US)
Pages (from-to)	5054-5056
Number of pages	3
Journal	Biochemistry
Volume	48
Issue number	23
DOIs	https://doi.org/10.1021/bi900164d
State	Published - Jun 16 2009

ASJC Scopus subject areas

Biochemistry

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abstract = "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 kcat/Km value for ATP-pyrophosphate exchange activity for D,L-N-acetylphosphinothricin was 3.5 μM-1 min-1, whereas the kcat/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).",

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T1 - In vitro characterization of a heterologously expressed nonribosomal peptide synthetase involved in phosphinothricin tripeptide biosynthesis

AU - Lee, Jin Hee

AU - Evans, Bradley S.

AU - Li, Gongyong

AU - Kelleher, Neil L.

AU - Van Der Donk, Wilfred A.

PY - 2009/6/16

Y1 - 2009/6/16

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AB - 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 kcat/Km value for ATP-pyrophosphate exchange activity for D,L-N-acetylphosphinothricin was 3.5 μM-1 min-1, whereas the kcat/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).

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In vitro characterization of a heterologously expressed nonribosomal peptide synthetase involved in phosphinothricin tripeptide biosynthesis

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