Probing a hydrogen bond pair and the FAD redox properties in the proline dehydrogenase domain of Escherichia coli PutA

The PutA flavoprotein from Escherichia coli combines DNA-binding, proline dehydrogenase (PRODH), and Δ¹-pyrroline-5-carboxylate dehydrogenase (P5CDH) activities onto a single polypeptide. Recently, an X-ray crystal structure of PutA residues 87-612 was solved which identified a D370-Y540 hydrogen bond pair in the PRODH active site that appears to have an important role in shaping proline binding and the FAD redox environment. To examine the role of D370-Y540 in the PRODH active site, mutants D370A, Y540F, and D370A/Y540F were characterized in a form of PutA containing only residues 86-601 (PutA86-601) designed to mimic the known structural region of PutA (87-612). Disruption of the D370-Y540 pair only slightly diminished k _cat, while more noticeable affects were observed in K_m. The mutant D370A/Y540F showed the most significant changes in the pH dependence of k_cat/K_m and K_m relative to wild-type PutA86-601 with an apparent pK_a value of about 8.2 for the pH-dependent decrease in K_m. From the pH profile of D370A/Y540F inhibition by L-tetrahydro-2-furoic acid (L-THFA), the pH dependency of K _m in D370A/Y540F is interpreted as resulting from the deprotonation of the proline amine in the E-S complex. Replacement of D370 and Y540 produces divergent effects on the E_m for bound FAD. At pH 7.0, E_m values of -0.026, -0.089 and -0.042 V were determined for the two-electron reduction of bound FAD in D370A, Y540F and D370A/Y540F, respectively. The 40-mV positive shift in E_m determined for D370A relative to wild-type PutA86-601 (E_m=-0.066 V, pH 7.0) indicates D370 has a key role in modulating the FAD redox environment.