title: Characterization of AusA: a dimodular nonribosomal peptide synthetase responsible for the production of aureusimine pyrazinones.
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.
authors.affiliation: Center for Drug Design, University of Minnesota , Minneapolis, MN 55455, United States.
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Wilson
authors.forename: Ce
authors.initials: C
authors.lastname: Shi
authors.forename: Aaron M
authors.initials: AM
authors.lastname: Teitelbaum
authors.forename: Andrew M
authors.initials: AM
authors.lastname: Gulick
authors.forename: Courtney C
authors.initials: CC
authors.lastname: Aldrich
mesh_terms: D003001:Cloning, Molecular; D007700:Kinetics; D010453:Peptide Synthases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011719:Pyrazines / Q000737:chemistry / Q000378:metabolism*; D013211:Staphylococcus aureus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D011719:Pyrazines; D010453:Peptide Synthases