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Biochemistry. 2013 Feb 5;52(5):926-37. doi: 10.1021/bi301330q. Epub 2013 Jan 23.

Characterization of AusA: a dimodular nonribosomal peptide synthetase responsible for the production of aureusimine pyrazinones.

Author information

1
Center for Drug Design, University of Minnesota , Minneapolis, MN 55455, United States.

Abstract

Aureusimines have been identified as potential virulence factors in Staphylococcus aureus. These pyrazinone secondary metabolites are produced by a nonribosomal peptide synthetase (NRPS) annotated as AusA. We report the overproduction of AusA as a 277 kDa soluble protein with A(1)-T(1)-C-A(2)-T(2)-R bimodular architecture. The substrate specificity of each adenylation (A) domain was initially probed using an ATP-pyrophosphate exchange assay with A-domain selective bisubstrate inhibitors to chemically knock out each companion A-domain. The activity of AusA was then reconstituted in vitro and shown to produce all naturally occurring aureusimines and non-natural pyrazinone products with k(cat) values ranging from 0.4 to 1.3 min(-1). Steady-state kinetic parameters were determined for all substrates and cofactors, providing the first comprehensive steady-state characterization of a NRPS employing a product formation assay. The K(M) values for the amino acids were up to 60-fold lower with the product formation assay than with the ATP-pyrophosphate exchange assay, most commonly used to assess A-domain substrate specificity. The C-terminal reductase (R) domain catalyzes reductive release of the dipeptidyl intermediate, leading to formation of an amino aldehyde that cyclizes to a dihydropyrazinone. We show oxidation to the final pyrazinone heterocycle is spontaneous. The activity and specificity of the R-domain was independently investigated using a NADPH consumption assay. AusA is a minimal autonomous two-module NRPS that represents an excellent model system for further kinetic and structural characterization.

PMID:
23302043
PMCID:
PMC3577359
DOI:
10.1021/bi301330q
[Indexed for MEDLINE]
Free PMC Article

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