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-γ-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. A 2.7 Å 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. 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.
Highlights
The very existence of proline utilization A (PutA) suggested the hypothesis that substrate channeling might occur in organisms where proline dehydrogenase (PRODH) and GSAL dehydrogenase (GSALDH) are distinct enzymes encoded by separate genes, as in eukaryotes and some bacteria
The available data suggest that communication between PRODH and GSALDH via substrate channeling is a conserved aspect of proline catabolism
The PRODH catalytic efficiency of CfPutA is on the low end for PutAs, which have reported values of 30 – 400 MϪ1 sϪ1 [4, 20, 22,23,24]
Summary
Proline Proline Proline P5C a Calculated from line of best fit over three data points (Fig. 3A). The diversity of PutAs extends to oligomeric state and quaternary structure. Type A PutAs form domain-swapped dimers [2, 6], and in at least one case (BjPutA), two of the dimers assemble into a ring-shaped tetramer [2] (Fig. 2C). Type C PutAs form a third distinct type of dimer, which is mediated by the DNA-binding domain [19]. We report the first structure of a class 3B PutA along with solution oligomeric state studies, kinetic characterization of substrate channeling, and domain deletion analysis probing the role of the C-terminal ALDHSF domain. The results further highlight the diversity of the PutA family and provide insight into structural basis of hysteretic substrate channeling
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