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

Abstract

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 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 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.

Determination of protein oligomeric structure from small‐angle X‐ray scattering

“…Thus, initial fitting to the experimental curves was carried out using only the tetramer model. The χ values obtained from fits to the crystallographic tetramer alone were in the range of 4.1–8.5, and there was a noticeable mismatch between the theoretical and experimental curves in both the Guinier region and the valley near q of 0.05–0.075 . This result indicated that the tetramer alone may not be sufficient to explain the experimental data.…”

“…The v values obtained from fits to the crystallographic tetramer alone were in the range of 4.1-8.5, and there was a noticeable mismatch between the theoretical and experimental curves in both the Guinier region and the valley near q of 0.05-0.075. 53 This result indicated that the tetramer alone may not be sufficient to explain the experimental data. To address this issue, MultiFoXS fitting was employed using all three models (dimer 1, dimer 2, and tetramer).…”

Structure, function, and mechanism of proline utilization A (PutA)

“…The GsPutA catalog includes complexes of the oxidized enzyme with proline analogs, structures of the enzyme in the 2-electron FAD reduced state, and a structure with the model electron acceptor menadione bisulfite (MB) bound in the PRODH active site. Also, the structure of another class 2A PutA was reported recently (PutA from Bdellovibrio bacteriovorus , BbPutA) [33]. …”

“…No other PutA studied to date forms this tetramer, so its functional significance is a matter of speculation. Recently, Korasick and coworkers showed that engineered dimeric forms of BjPutA retain full catalytic activity, suggesting that tetramerization is not essential for in vitro activity [33]. Whether the tetramer is needed for in vivo function remains to be studied.…”

Discovery of the Membrane Binding Domain in Trifunctional Proline Utilization A

“…PutAs in the type A class contain the two catalytic domains and are the best structurally characterized with high-resolution crystal structures of PutA from Bradyrhizobium japonicum ( Bj PutA), 27 Geobacter sulfurreducens ( Gs PutA), 28 and Bdellovibrio bacteriovorus . 34 Type B PutAs are distinguished by an additional C-terminal domain that was recently identified as an aldehyde dehydrogenase superfamily (ALDHSF) fold. Characterization of type B PutAs from Sinorhizobium meliloti 29 and Corynebacteirum freiburgense 35 showed that the major function of the C-terminal ALDHSF domain is to stabilize aldehyde binding in the GSALDH active site and seal the substrate channel from bulk solvent.…”