Kinetic Analysis of Three Activated Phenylalanyl Intermediates Generated by the Initiation Module PheATE of Gramicidin S Synthetase

Luo, Lusong; Walsh, Christopher T.

doi:10.1021/bi015518+

Cited by 46 publications

(81 citation statements)

References 27 publications

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“…Next, we investigated the prototypical nonribosomal peptide synthetase GrsA involved in biosynthesis of gramicidin that contains the adenylation domain and thiolation domain on the same polypeptide 21, 29. Previous attempts to functionally characterize the adenylation activity of the phenylalanine adenylation domain have relied on the ATP-PP i exchange assay, since the carrier domain is located in cis precluding steady-state kinetic analysis in the overall forward direction.…”

Section: Resultsmentioning

confidence: 99%

A continuous kinetic assay for adenylation enzyme activity and inhibition

Wilson

Aldrich

2010

Analytical Biochemistry

148

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Adenylation/adenylate-forming enzymes catalyze the activation of a carboxylic acid at the expense of ATP to form an acyl-adenylate intermediate and pyrophosphate (PP i ). In a second half-reaction, adenylation enzymes catalyze the transfer of the acyl moiety of the acyl-adenylate onto an acceptor molecule, which can be either a protein or a small molecule. We describe the design, development, and validation of a coupled continuous spectrophotometric assay for adenylation enzymes that employs hydroxylamine as a surrogate acceptor molecule leading to the formation of a hydroxamate. The released pyrophosphate from the first half-reaction is measured using the pyrophosphatasepurine nucleoside phosphorylase coupling system with the chromogenic substrate 7-methylthioguanosine (MesG). The coupled hydroxamate-MesG assay is especially useful for characterizing the activity and inhibition of adenylation enzymes that acylate a protein substrate and/ or fail to undergo rapid ATP-PP i exchange.

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Section: Resultsmentioning

confidence: 99%

A continuous kinetic assay for adenylation enzyme activity and inhibition

Wilson

Aldrich

2010

Analytical Biochemistry

148

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“…[33][34][35][36] We and others evaluated mechanisms in lipopeptide biosynthesis for loading of acyl chains onto the free amine of the aminoacyl 1 -S-module 1. [33][34][35][36] We and others evaluated mechanisms in lipopeptide biosynthesis for loading of acyl chains onto the free amine of the aminoacyl 1 -S-module 1.…”

Section: Chain Initiationmentioning

confidence: 99%

Insights into the chemical logic and enzymatic machinery of NRPS assembly lines

2016

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Appreciation that some cyclic peptide antibiotics such as gramicidin S and tyrocidine were nonribosomally synthesized has been known for 50 years. The past two decades of research including advances in bacterial genetics, genomics, protein biochemistry and mass spectrometry have codified the principles of assembly line enzymology for hundreds of nonribosomal peptides and in parallel for thousands of polyketides. The advances in understanding the strategies used for chain initiation, elongation and termination from these assembly lines have revitalized natural product biosynthetic communities.

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“…Racemization takes place after the activation of the amino acid on an enzymeControl of Fluxes Towards Antibiotics and the Role of Primary Metabolism 163 Fig. 14 for phenylalanine in the cyclic decapeptide gramicidin S [74][75][76].…”

Section: The Role Of Synthesis Of Specific Precursorsmentioning

confidence: 99%