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

Journal Article · · Federation of European Biochemical Societies (FEBS) Journal
DOI:https://doi.org/10.1111/febs.14165· OSTI ID:1409112
 [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

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:
Argonne National Laboratory (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Organization:
DOE - BASIC ENERGY SCIENCESNIH
OSTI ID:
1409112
Journal Information:
Federation of European Biochemical Societies (FEBS) Journal, Vol. 284, Issue 18; ISSN 1742-464X
Publisher:
Federation of European Biochemical Societies
Country of Publication:
United States
Language:
ENGLISH

References (59)

SASBDB, a repository for biological small-angle scattering data journal October 2014
Uniqueness of ab initio shape determination in small-angle scattering journal April 2003
Impact of disease-Linked mutations targeting the oligomerization interfaces of aldehyde dehydrogenase 7A1 journal October 2017
Crystal structure of the bifunctional proline utilization A flavoenzyme from Bradyrhizobium japonicum journal February 2010
Hot spots-A review of the protein-protein interface determinant amino-acid residues journal June 2007
DAMMIF, a program for rapid ab-initio shape determination in small-angle scattering journal January 2009
Structures of the Escherichia coli PutA Proline Dehydrogenase Domain in Complex with Competitive Inhibitors , journal October 2004
Determination of the regularization parameter in indirect-transform methods using perceptual criteria journal August 1992
Anatomy of hot spots in protein interfaces journal July 1998
PRIMUS: a Windows PC-based system for small-angle scattering data analysis journal September 2003
The Proline Regulatory Axis and Cancer journal January 2012
Structure-based model of allostery predicts coupling between distant sites journal March 2012
Regulation of proline metabolism in mycobacteria and its role in carbon metabolism under hypoxia: Proline metabolism in Mycobacterium journal April 2012
Structure, Dynamics, Assembly, and Evolution of Protein Complexes journal June 2015
Effects of proline analog binding on the spectroscopic and redox properties of PutA journal December 2002
Structural Aspects of Aldehyde Dehydrogenase that Influence Dimer−Tetramer Formation journal July 2002
MolProbity: all-atom contacts and structure validation for proteins and nucleic acids journal May 2007
Structural Basis for the Inactivation of Thermus thermophilus Proline Dehydrogenase by N- Propargylglycine , journal May 2008
Using Situs for the integration of multi-resolution structures journal January 2010
An approach to multi-copy search in molecular replacement journal December 2000
FoXS, FoXSDock and MultiFoXS: Single-state and multi-state structural modeling of proteins and their complexes based on SAXS profiles journal May 2016
Size-Distribution Analysis of Macromolecules by Sedimentation Velocity Ultracentrifugation and Lamm Equation Modeling journal March 2000
Structural Symmetry and Protein Function journal June 2000
Scaling and assessment of data quality journal December 2005
Inference of Macromolecular Assemblies from Crystalline State journal September 2007
XDS journal January 2010
Phylogeny.fr: robust phylogenetic analysis for the non-specialist journal May 2008
Substrate channeling in proline metabolism journal January 2012
Ehrlichia chaffeensis Proliferation Begins with NtrY/NtrX and PutA/GlnA Upregulation and CtrA Degradation Induced by Proline and Glutamine Uptake journal November 2014
Robust, high-throughput solution structural analyses by small angle X-ray scattering (SAXS) journal July 2009
Structure and characterization of a class 3B proline utilization A: Ligand-induced dimerization and importance of the C-terminal domain for catalysis journal July 2017
Reactive Oxygen Species Homeostasis and Virulence of the Fungal Pathogen Cryptococcus neoformans Requires an Intact Proline Catabolism Pathway journal April 2013
The Structure of the Proline Utilization A Proline Dehydrogenase Domain Inactivated by N -Propargylglycine Provides Insight into Conformational Changes Induced by Substrate Binding and Flavin Reduction , journal January 2010
Evidence for Hysteretic Substrate Channeling in the Proline Dehydrogenase and Δ 1 -Pyrroline-5-carboxylate Dehydrogenase Coupled Reaction of Proline Utilization A (PutA) journal December 2013
Kinetic and Structural Characterization of Tunnel-Perturbing Mutants in Bradyrhizobium japonicum Proline Utilization A journal July 2014
Understanding allosteric and cooperative interactions in enzymes journal September 2013
Implementation and performance of SIBYLS: a dual endstation small-angle X-ray scattering and macromolecular crystallography beamline at the Advanced Light Source journal January 2013
RaptorX server: A Resource for Template-Based Protein Structure Modeling book January 2014
Evidence That the C-Terminal Domain of a Type B PutA Protein Contributes to Aldehyde Dehydrogenase Activity and Substrate Channeling journal August 2014
Crystal Structures and Kinetics of Monofunctional Proline Dehydrogenase Provide Insight into Substrate Recognition and Conformational Changes Associated with Flavin Reduction and Product Release journal December 2012
MolProbity : all-atom structure validation for macromolecular crystallography journal December 2009
FoXS: a web server for rapid computation and fitting of SAXS profiles journal May 2010
Resolving the cofactor-binding site in the proline biosynthetic enzyme human pyrroline-5-carboxylate reductase 1 journal April 2017
Features and development of Coot journal March 2010
Structures of the PutA peripheral membrane flavoenzyme reveal a dynamic substrate-channeling tunnel and the quinone-binding site journal February 2014
PRODH variants and risk for schizophrenia journal June 2008
Solvent content of protein crystals journal April 1968
PHENIX: a comprehensive Python-based system for macromolecular structure solution journal January 2010
Possible involvement of put A gene in Helicobacter pylori colonization in the stomach and motility journal January 2008
Automated matching of high- and low-resolution structural models journal February 2001
Fast, scalable generation of high‐quality protein multiple sequence alignments using Clustal Omega journal January 2011
Determination of the molecular weight of proteins in solution from a single small-angle X-ray scattering measurement on a relative scale journal December 2009
Morpheeins – a new structural paradigm for allosteric regulation journal September 2005
SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information journal April 2014
Protein structure prediction on the Web: a case study using the Phyre server journal February 2009
Structural biology of proline catabolism journal March 2008
Accurate assessment of mass, models and resolution by small-angle scattering journal April 2013
Structural Determinants of Oligomerization of Δ1-Pyrroline-5-Carboxylate Dehydrogenase: Identification of a Hexamerization Hot Spot journal September 2013
Genetic variation at the 22q11 PRODH2/DGCR6 locus presents an unusual pattern and increases susceptibility to schizophrenia journal March 2002