Biophysical investigation of type A PutAs reveals a conserved core oligomeric structure.

Many enzymes form homooligomers, yet the functional significance of self-association is seldom obvious. Herein, we examine the connection between oligomerization and catalytic function for proline utilization A (PutA) enzymes. PutAs are bifunctional enzymes that catalyze both reactions of proline catabolism. Type A PutAs are the smallest members of the family, possessing a minimal domain architecture consisting of N-terminal proline dehydrogenase and C-terminal l-glutamate-γ-semialdehyde dehydrogenase modules. Type A PutAs form domain-swapped dimers, and in one case ( Bradyrhizobium japonicum PutA), two of the dimers assemble into a ring-shaped tetramer. Whereas the dimer has a clear role in substrate channeling, the functional significance of the tetramer is unknown. To address this question, we performed structural studies of four-type A PutAs from two clades of the PutA tree. The crystal structure of Bdellovibrio bacteriovorus PutA covalently inactivated by N-propargylglycine revealed a fold and substrate-channeling tunnel similar to other PutAs. Small-angle X-ray scattering ( SAXS) and analytical ultracentrifugation indicated that Bdellovibrio PutA is dimeric in solution, in contrast to the prediction from crystal packing of a stable tetrameric assembly. SAXS studies of two other type A PutAs from separate clades also suggested that the dimer predominates in solution. To assess whether the tetramer of B. japonicum PutA is necessary for catalytic function, a hot spot disruption mutant that cleanly produces dimeric protein was generated. The dimeric variant exhibited kinetic parameters similar to the wild-type enzyme. These results implicate the domain-swapped dimer as the core structural and functional unit of type A PutAs. Enzymes Proline dehydrogenase (); l-glutamate-γ-semialdehyde dehydrogenase (). Databases The atomic coordinates and structure factor amplitudes have been deposited in the Protein Data Bank under accession number . The SAXS data have been deposited in the SASBDB under the following accession codes: SASDCP3 (BbPutA), SASDCQ3 (DvPutA 1.5 mg·mL−1), SASDCX3 (DvPutA 3.0 mg·mL−1), SASDCY3 (DvPutA 4.5 mg·mL−1), SASDCR3 (LpPutA 3.0 mg·mL−1), SASDCV3 (LpPutA 5.0 mg·mL−1), SASDCW3 (LpPutA 8.0 mg·mL−1), SASDCS3 (BjPutA 2.3 mg·mL−1), SASDCT3 (BjPutA 4.7 mg·mL−1), SASDCU3 (BjPutA 7.0 mg·mL−1), SASDCZ3 (R51E 2.3 mg·mL−1), SASDC24 (R51E 4.7 mg·mL−1), SASDC34 (R51E 7.0 mg·mL−1). [ABSTRACT FROM AUTHOR]