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

Korasick, David A.; Singh, Harkewal; Pemberton, Travis A.; Luo, Min; Dhatwalia, Richa; Tanner, John J.

doi:10.1111/febs.14165

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

Journal Article · Tue Aug 01 04:00:00 EDT 2017 · Federation of European Biochemical Societies (FEBS) Journal

DOI:https://doi.org/10.1111/febs.14165· OSTI ID:1409112

Korasick, David A. ^[1]; Singh, Harkewal ^[2]; Pemberton, Travis A. ^[2]; Luo, Min ^[2]; Dhatwalia, Richa ^[2]; Tanner, John J. ^[3]

Department of Biochemistry, University of Missouri, Columbia MO USA
Department of Chemistry, University of Missouri, Columbia MO USA
Department of Biochemistry, University of Missouri, Columbia MO USA; Department of Chemistry, University of Missouri, Columbia MO USA

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.

Research Organization:: Advanced Photon Source (APS), Argonne National Laboratory (ANL), Argonne, IL (US)

Sponsoring Organization:: DOE - BASIC ENERGY SCIENCESNIH

OSTI ID:: 1409112

Journal Information:: Federation of European Biochemical Societies (FEBS) Journal, Journal Name: Federation of European Biochemical Societies (FEBS) Journal Journal Issue: 18 Vol. 284; ISSN 1742-464X

Publisher:: Federation of European Biochemical Societies

Country of Publication:: United States

Language:: ENGLISH

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