Structure and characterization of a class 3B proline utilization A: Ligand-induced dimerization and importance of the C-terminal domain for catalysis

The bifunctional flavoenzyme proline utilization A (PutA) catalyzes the two-step oxidation of proline to glutamate using separate proline dehydrogenase (PRODH) and L-glutamate-semialdehyde dehydrogenase active sites. Because PutAs catalyze sequential reactions, they are good systems for studying how metabolic enzymes communicate via substrate channeling. Although mechanistically similar, PutAs vary widely in domain architecture, oligomeric state, and quaternary structure, and these variations represent different structural solutions to the problem of sequestering a reactive metabolite. Here, we studied PutA from Corynebacterium freiburgense (CfPutA), which belongs to the uncharacterized 3B class of PutAs.A2.7Å resolution crystal structure showed the canonical arrangement of PRODH, L-glutamate- γ-semialdehyde dehydrogenase, and C-terminal domains, including an extended interdomain tunnel associated with substrate channeling.Thestructure unexpectedly revealed a novelopenconformation of thePRODHactive site, which is interpreted to represent the non-activated conformation, an elusive form of PutA that exhibits suboptimal channeling. Nevertheless, CfPutA exhibited normal substrate-channeling activity, indicating that it isomerizes into the active state under assay conditions. Sedimentation-velocity experiments provided insight into the isomerization process, showing that CfPutA dimerizes in the presence of a proline analog andNAD. These results are consistent with the morpheein model of enzyme hysteresis, in which substrate binding induces conformational changes that promote assembly of a high-activity oligomer. Finally, we used domain deletion analysis to investigate the function of the C-terminal domain. Although this domain contains neither catalytic residues nor substrate sites, its removal impaired both catalytic activities, suggesting that it may be essential for active-site integrity.