Domain Communication in Bifunctional Proline Utilization A from Sinorhizobium Meliloti

The reduction potential of flavin bound to Sinorhizobium meliloti proline utilization A (SmPutA) is beneficial in providing vital information on enzyme activity and the active site environment. Flavin is crucial in biological metabolism such as aerobic respiration and other important processes like oxygen activation that are catalyzed by flavin-dependent enzymes. (4). In this study, we seek to determine whether the redox potential of SmPutA-bound flavin can be used to characterize potential long-distance communication and analyze the possibilities of inactivation reversibility. SmPutA is a large bifunctional enzyme that catalyzes the overall oxidation of proline to glutamate via two catalytic domains (1). The first domain is proline dehydrogenase (PRODH) which catalyzes the flavin-dependent oxidation of L-proline to Δ1-pyrroline-5-carboxylate (P5C) (1). The second domain catalyzes the NAD+-dependent oxidation of L-glutamic-γ-semialdehyde (GSAL) to L-glutamate (1). The overall reaction is facilitated by 40 Å tunnel that connects the two catalytic domains enabling P5C to travel directly from the PRODH domain to the GSAL dehydrogenase (GSALDH) domain (1). Here we explore long-range communication between the PRODH and GSALDH domains using redox potentiometry. The redox potential of the flavin bound in the PRODH domain was measured with different ligands bound to the GSALDH site. The reversibility of the inhibiting ligand, Ebselen, bound to the GSALDH site was also studied for activity recovery. Discovering a change in the flavin redox potential upon ligand binding and reversing inactivation would provide novel insights into large-scale conformational changes in SmPutA to enhance substrate channeling and the understanding of PutA function.