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Title: 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 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. japonicummore » 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.« less

Authors:
 [1];  [2];  [2];  [2];  [2];  [3]
  1. Department of Biochemistry, University of Missouri, Columbia MO USA
  2. Department of Chemistry, University of Missouri, Columbia MO USA
  3. Department of Biochemistry, University of Missouri, Columbia MO USA; Department of Chemistry, University of Missouri, Columbia MO USA
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Org.:
DOE - BASIC ENERGY SCIENCESNIH
OSTI Identifier:
1409112
Resource Type:
Journal Article
Journal Name:
Federation of European Biochemical Societies (FEBS) Journal
Additional Journal Information:
Journal Volume: 284; Journal Issue: 18; Journal ID: ISSN 1742-464X
Publisher:
Federation of European Biochemical Societies
Country of Publication:
United States
Language:
ENGLISH
Subject:
59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Korasick, David A., Singh, Harkewal, Pemberton, Travis A., Luo, Min, Dhatwalia, Richa, and Tanner, John J. Biophysical investigation of type A PutAs reveals a conserved core oligomeric structure. United States: N. p., 2017. Web. doi:10.1111/febs.14165.
Korasick, David A., Singh, Harkewal, Pemberton, Travis A., Luo, Min, Dhatwalia, Richa, & Tanner, John J. Biophysical investigation of type A PutAs reveals a conserved core oligomeric structure. United States. doi:10.1111/febs.14165.
Korasick, David A., Singh, Harkewal, Pemberton, Travis A., Luo, Min, Dhatwalia, Richa, and Tanner, John J. Tue . "Biophysical investigation of type A PutAs reveals a conserved core oligomeric structure". United States. doi:10.1111/febs.14165.
@article{osti_1409112,
title = {Biophysical investigation of type A PutAs reveals a conserved core oligomeric structure},
author = {Korasick, David A. and Singh, Harkewal and Pemberton, Travis A. and Luo, Min and Dhatwalia, Richa and Tanner, John J.},
abstractNote = {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.},
doi = {10.1111/febs.14165},
journal = {Federation of European Biochemical Societies (FEBS) Journal},
issn = {1742-464X},
number = 18,
volume = 284,
place = {United States},
year = {2017},
month = {8}
}

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