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

Authors

David A. Korasick, University of Missouri
Thameesha T. Gamage, University of Missouri
Shelbi Christgen, University of Nebraska - LincolnFollow
Kyle M. Stiers, University of Missouri
Lesa J. Beamer, University of Missouri
Michael T. Henzl, University of Missouri
Donald F. Becker, University of Nebraska-LincolnFollow
John J. Tanner, University of MissouriFollow

Date of this Version

2017

Citation

J. Biol. Chem. (2017) 292(23) 9652–9665

Comments

© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

DOI 10.1074/jbc.M117.786855

Abstract

The bifunctional flavoenzyme proline utilization A (PutA) catalyzes the two-step oxidation of proline to glutamate using separate proline dehydrogenase (PRODH) and L-glutamate-y-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.A2.7A˚ resolution crystal structure showed the canonical arrangement of PRODH, L-glutamate-y-semialdehyde dehydrogenase, and C-terminal domains, including an extended interdomain tunnel associated with substrate channeling. The structure unexpectedly revealed a novel open conformation of the PRODH active 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 and NAD⁺. 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.