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 A1-T 1-C-A2-T2-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 kcat 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 KM 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.