Figure 4.

Recombinant GST-NadB possesses l-aspartate oxidase activity that is abolished by mutation of Arg²⁷⁵ to Leu. The graph depicts the second step of the two-step assay, demonstrating the change in absorbance at 340 nm resulting from oxidation of NADH and reduction of OAA in the presence of commercial malate dehydrogenase, if the first step of the assay produced iminoaspartate (which spontaneously hydrolyzes to OAA). A decrease in absorbance will only occur if the protein used in the first step possesses l-aspartate oxidase activity. Proteins used in the first step were GST-NadB (circles), GST (squares), and GST-R275L-NadB (triangles). The graph represents the mean ± S.D. of three independent experiments.