FIG. 1.

Recombinant N-His₆-GpsA protein is an active G3P dehydrogenase in vitro. R. prowazekii gpsA was cloned into pET15b, heterologously expressed in E. coli BL21(DE3), and purified with N-His₆-GpsA. (A) Purified protein was resolved on a 4 to 15% Tris-HCl SDS-polyacrylamide gel and visualized by Imperial protein staining and Western blot analysis using anti-His₆ monoclonal antibody (sizes [in kDa] are indicated on the left). The mass spectrometry analysis coverage map shows matched peptides in bold font (30% coverage). (B) N-His₆-GpsA-catalyzed conversion of [³²P]DHAP to [³²P]G3P was verified by paper chromatography. [³²P]ATP, [³²P]DHAP, [³²P]G3P, and [³²P]orthophosphate were used as standards. The reaction mixtures were incubated for 60 min at room temperature prior to chromatographic analysis. Lane A, a negative-control reaction mixture containing 6 μM [³²P]DHAP and 100 μM NADPH only; lane B, a negative-control reaction mixture containing [³²P]DHAP and 2.5 μg ml⁻¹ N-His₆-GpsA protein only; lane C, the N-His₆-GpsA-catalyzed conversion of [³²P]DHAP to [³²P]G3P absolutely required the presence of NADPH. The density of each compound on the chromatograph is expressed as a percentage of the total lane density (summarized in tabular form to the right of each spot on the chromatograph). (C) N-His₆-GpsA-catalyzed conversion of DHAP to G3P was measured spectrophotometrically at OD₃₄₀ by following the oxidation of NADPH or NADH over time. Error bars represent standard deviations. The reaction mixtures contained 1.0 μg ml⁻¹ N-His₆-GpsA, 500 μM DHAP, and 100 μM NAD(P)H in modified buffer A; and the reactions were carried out at room temperature.