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Title: The structure of Medicago truncatula δ 1-pyrroline-5-carboxylate reductase provides new insights into regulation of proline biosynthesis in plants

Abstract

The two pathways for proline biosynthesis in higher plants share the last step, the conversion of δ 1-pyrroline-5-carboxylate (P5C) to L-proline, which is catalyzed by P5C reductase (P5CR, EC 1.5.1.2) with the use of NAD(P)H as a coenzyme. There is increasing amount of evidence to suggest a complex regulation of P5CR activity at the post-translational level, yet the molecular basis of these mechanisms is unknown. Here we report the three-dimensional structure of the P5CR enzyme from the model legume Medicago truncatula (Mt). The crystal structures of unliganded MtP5CR decamer, and its complexes with the products NAD +, NADP +, and L-proline were refined using x-ray diffraction data (at 1.7, 1.85, 1.95, and 2.1 Å resolution, respectively). Based on the presented structural data, the coenzyme preference for NADPH over NADH was explained, and NADPH is suggested to be the only coenzyme used by MtP5CR in vivo. Moreover, the insensitivity of MtP5CR to feed-back inhibition by proline, revealed by enzymatic analysis, was correlated with structural features. Additionally, a mechanism for the modulation of enzyme activity by chloride anions is discussed, as well as the rationale for the possible development of effective enzyme inhibitors.

Authors:
 [1];  [2];  [3];  [1]
  1. National Cancer Institute, Argonne, IL (United States)
  2. Argonne National Lab. (ANL), Argonne, IL (United States)
  3. Univ. of Ferrara, Ferrara (Italy)
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States)
Sponsoring Org.:
USDOE Office of Science (SC)
OSTI Identifier:
1239950
Grant/Contract Number:  
AC02-06CH11357
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
Frontiers in Plant Science
Additional Journal Information:
Journal Volume: 6; Journal ID: ISSN 1664-462X
Publisher:
Frontiers Research Foundation
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; protein structure; decamer; coenzyme preference; salt stress; abiotic stress; P5C reductase; P5CR

Citation Formats

Ruszkowski, Milosz, Nocek, Boguslaw, Forlani, Giuseppe, and Dauter, Zbigniew. The structure of Medicago truncatula δ1-pyrroline-5-carboxylate reductase provides new insights into regulation of proline biosynthesis in plants. United States: N. p., 2015. Web. doi:10.3389/fpls.2015.00869.
Ruszkowski, Milosz, Nocek, Boguslaw, Forlani, Giuseppe, & Dauter, Zbigniew. The structure of Medicago truncatula δ1-pyrroline-5-carboxylate reductase provides new insights into regulation of proline biosynthesis in plants. United States. doi:10.3389/fpls.2015.00869.
Ruszkowski, Milosz, Nocek, Boguslaw, Forlani, Giuseppe, and Dauter, Zbigniew. Fri . "The structure of Medicago truncatula δ1-pyrroline-5-carboxylate reductase provides new insights into regulation of proline biosynthesis in plants". United States. doi:10.3389/fpls.2015.00869. https://www.osti.gov/servlets/purl/1239950.
@article{osti_1239950,
title = {The structure of Medicago truncatula δ1-pyrroline-5-carboxylate reductase provides new insights into regulation of proline biosynthesis in plants},
author = {Ruszkowski, Milosz and Nocek, Boguslaw and Forlani, Giuseppe and Dauter, Zbigniew},
abstractNote = {The two pathways for proline biosynthesis in higher plants share the last step, the conversion of δ1-pyrroline-5-carboxylate (P5C) to L-proline, which is catalyzed by P5C reductase (P5CR, EC 1.5.1.2) with the use of NAD(P)H as a coenzyme. There is increasing amount of evidence to suggest a complex regulation of P5CR activity at the post-translational level, yet the molecular basis of these mechanisms is unknown. Here we report the three-dimensional structure of the P5CR enzyme from the model legume Medicago truncatula (Mt). The crystal structures of unliganded MtP5CR decamer, and its complexes with the products NAD+, NADP+, and L-proline were refined using x-ray diffraction data (at 1.7, 1.85, 1.95, and 2.1 Å resolution, respectively). Based on the presented structural data, the coenzyme preference for NADPH over NADH was explained, and NADPH is suggested to be the only coenzyme used by MtP5CR in vivo. Moreover, the insensitivity of MtP5CR to feed-back inhibition by proline, revealed by enzymatic analysis, was correlated with structural features. Additionally, a mechanism for the modulation of enzyme activity by chloride anions is discussed, as well as the rationale for the possible development of effective enzyme inhibitors.},
doi = {10.3389/fpls.2015.00869},
journal = {Frontiers in Plant Science},
issn = {1664-462X},
number = ,
volume = 6,
place = {United States},
year = {2015},
month = {10}
}

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Works referenced in this record:

Arginine Catabolism by Microorganisms
journal, October 1979


Gapped BLAST and PSI-BLAST: a new generation of protein database search programs
journal, September 1997

  • Altschul, Stephen F.; Madden, Thomas L.; Schäffer, Alejandro A.
  • Nucleic Acids Research, Vol. 25, Issue 17, p. 3389-3402
  • DOI: 10.1093/nar/25.17.3389

Transcriptional regulation of proline biosynthesis in Medicago truncatula reveals developmental and environmental specific features
journal, March 2004


Cellular and whole-plant chloride dynamics in barley: insights into chloride?nitrogen interactions and salinity responses
journal, February 2004


Prokaryotic Osmoregulation: Genetics and Physiology
journal, October 1991


Exogenous ornithine is an effective precursor and the δ-ornithine amino transferase pathway contributes to proline accumulation under high N recycling in salt-stressed cashew leaves
journal, January 2012

  • da Rocha, Iza Marineves Almeida; Vitorello, Victor Alexandre; Silva, Jamille Santos
  • Journal of Plant Physiology, Vol. 169, Issue 1
  • DOI: 10.1016/j.jplph.2011.08.001

PDBsum additions
journal, October 2013

  • de Beer, Tjaart A. P.; Berka, Karel; Thornton, Janet M.
  • Nucleic Acids Research, Vol. 42, Issue D1
  • DOI: 10.1093/nar/gkt940

Proline biosynthesis and osmoregulation in plants
journal, August 1993


The Role of Δ 1 -Pyrroline-5-Carboxylate Dehydrogenase in Proline Degradation
journal, November 2004

  • Deuschle, Karen; Funck, Dietmar; Forlani, Giuseppe
  • The Plant Cell, Vol. 16, Issue 12
  • DOI: 10.1105/tpc.104.023622

Improved R-factors for diffraction data analysis in macromolecular crystallography
journal, April 1997

  • Diederichs, Kay; Karplus, P. Andrew
  • Nature Structural Biology, Vol. 4, Issue 4
  • DOI: 10.1038/nsb0497-269

Features and development of Coot
journal, March 2010

  • Emsley, P.; Lohkamp, B.; Scott, W. G.
  • Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 4
  • DOI: 10.1107/S0907444910007493

New LIC vectors for production of proteins from genes containing rare codons
journal, September 2013

  • Eschenfeldt, William H.; Makowska-Grzyska, Magdalena; Stols, Lucy
  • Journal of Structural and Functional Genomics, Vol. 14, Issue 4
  • DOI: 10.1007/s10969-013-9163-9

Proline Accumulation and AtP5CS2 Gene Activation Are Induced by Plant-Pathogen Incompatible Interactions in Arabidopsis
journal, April 2004

  • Fabro, Georgina; Kovács, Izabella; Pavet, Valeria
  • Molecular Plant-Microbe Interactions, Vol. 17, Issue 4
  • DOI: 10.1094/MPMI.2004.17.4.343

Synthesis and Evaluation of Effective Inhibitors of Plant δ 1 -Pyrroline-5-carboxylate Reductase
journal, July 2013

  • Forlani, Giuseppe; Berlicki, Łukasz; Duò, Mattia
  • Journal of Agricultural and Food Chemistry, Vol. 61, Issue 28
  • DOI: 10.1021/jf401234s

Functional properties and structural characterization of rice δ1-pyrroline-5-carboxylate reductase
journal, July 2015

  • Forlani, Giuseppe; Bertazzini, Michele; Zarattini, Marco
  • Frontiers in Plant Science, Vol. 6
  • DOI: 10.3389/fpls.2015.00565

Plant P5C Reductase as a New Target for Aminomethylenebisphosphonates
journal, May 2007

  • Forlani, Giuseppe; Giberti, Samuele; Berlicki, Łukasz
  • Journal of Agricultural and Food Chemistry, Vol. 55, Issue 11
  • DOI: 10.1021/jf0701032

Evolution of plant δ1-pyrroline-5-carboxylate reductases from phylogenetic and structural perspectives
journal, August 2015

  • Forlani, Giuseppe; Makarova, Kira S.; Ruszkowski, Milosz
  • Frontiers in Plant Science, Vol. 6
  • DOI: 10.3389/fpls.2015.00567

Tailoring the Structure of Aminobisphosphonates To Target Plant P5C Reductase
journal, May 2008

  • Forlani, Giuseppe; Occhipinti, Andrea; Berlicki, Łukasz
  • Journal of Agricultural and Food Chemistry, Vol. 56, Issue 9
  • DOI: 10.1021/jf800029t

δ1-Pyrroline-5-carboxylate reductase as a new target for therapeutics: inhibition of the enzyme from Streptococcus pyogenes and effects in vivo
journal, July 2011


[delta]1-Pyrroline-5-Carboxylate Dehydrogenase from Cultured Cells of Potato (Purification and Properties)
journal, April 1997


Requirement of proline synthesis during Arabidopsis reproductive development
journal, January 2012


Proline antagonizes GABA-induced quenching of quorum-sensing in Agrobacterium tumefaciens
journal, August 2009

  • Haudecoeur, E.; Planamente, S.; Cirou, A.
  • Proceedings of the National Academy of Sciences, Vol. 106, Issue 34
  • DOI: 10.1073/pnas.0808005106

Enhanced dihydroflavonol-4-reductase activity and NAD homeostasis leading to cell death tolerance in transgenic rice
journal, April 2005

  • Hayashi, M.; Takahashi, H.; Tamura, K.
  • Proceedings of the National Academy of Sciences, Vol. 102, Issue 19
  • DOI: 10.1073/pnas.0502556102

NADH-dependent metabolism of nitric oxide in alfalfa root cultures expressing barley hemoglobin
journal, May 2004


XDS
journal, January 2010

  • Kabsch, Wolfgang
  • Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 2
  • DOI: 10.1107/S0907444909047337

Dictionary of protein secondary structure: Pattern recognition of hydrogen-bonded and geometrical features
journal, December 1983


High-throughput protein purification and quality assessment for crystallization
journal, September 2011


Activities of the Pentose Phosphate Pathway and Enzymes of Proline Metabolism in Legume Root Nodules
journal, November 1990

  • Kohl, Daniel H.; Lin, Jih-Jing; Shearer, Georgia
  • Plant Physiology, Vol. 94, Issue 3
  • DOI: 10.1104/pp.94.3.1258

Proline metabolism in N2-fixing root nodules: energy transfer and regulation of purine synthesis.
journal, April 1988

  • Kohl, D. H.; Schubert, K. R.; Carter, M. B.
  • Proceedings of the National Academy of Sciences, Vol. 85, Issue 7
  • DOI: 10.1073/pnas.85.7.2036

ACHESYM : an algorithm and server for standardized placement of macromolecular models in the unit cell
journal, November 2014

  • Kowiel, Marcin; Jaskolski, Mariusz; Dauter, Zbigniew
  • Acta Crystallographica Section D Biological Crystallography, Vol. 70, Issue 12
  • DOI: 10.1107/S1399004714024572

Automated macromolecular model building for X-ray crystallography using ARP/wARP version 7
journal, June 2008

  • Langer, Gerrit; Cohen, Serge X.; Lamzin, Victor S.
  • Nature Protocols, Vol. 3, Issue 7
  • DOI: 10.1038/nprot.2008.91

Proline metabolism and transport in plant development
journal, March 2010


Structure validation by Cα geometry: ϕ,ψ and Cβ deviation
journal, January 2003

  • Lovell, Simon C.; Davis, Ian W.; Arendall, W. Bryan
  • Proteins: Structure, Function, and Bioinformatics, Vol. 50, Issue 3
  • DOI: 10.1002/prot.10286

Proline accumulation in plants: Not only stress
journal, November 2009

  • Mattioli, Roberto; Costantino, Paolo; Trovato, Maurizio
  • Plant Signaling & Behavior, Vol. 4, Issue 11
  • DOI: 10.4161/psb.4.11.9797

Modulation of intracellular proline levels affects flowering time and inflorescence architecture in Arabidopsis
journal, December 2007

  • Mattioli, Roberto; Marchese, Daniele; D’Angeli, Simone
  • Plant Molecular Biology, Vol. 66, Issue 3
  • DOI: 10.1007/s11103-007-9269-1

Phaser crystallographic software
journal, July 2007

  • McCoy, Airlie J.; Grosse-Kunstleve, Ralf W.; Adams, Paul D.
  • Journal of Applied Crystallography, Vol. 40, Issue 4
  • DOI: 10.1107/S0021889807021206

Crystal Structure of Human Pyrroline-5-carboxylate Reductase
journal, June 2006


HKL -3000: the integration of data reduction and structure solution – from diffraction images to an initial model in minutes
journal, July 2006

  • Minor, Wladek; Cymborowski, Marcin; Otwinowski, Zbyszek
  • Acta Crystallographica Section D Biological Crystallography, Vol. 62, Issue 8
  • DOI: 10.1107/S0907444906019949

Purification and Characterization of Δ1-Pyrroline-5-Carboxylate Reductase Isoenzymes, Indicating Differential Distribution in Spinach (Spinacia oleracea L.) Leaves
journal, July 2001

  • Murahama, Minoru; Yoshida, Takayuki; Hayashi, Fumio
  • Plant and Cell Physiology, Vol. 42, Issue 7
  • DOI: 10.1093/pcp/pce093

REFMAC 5 for the refinement of macromolecular crystal structures
journal, March 2011

  • Murshudov, Garib N.; Skubák, Pavol; Lebedev, Andrey A.
  • Acta Crystallographica Section D Biological Crystallography, Vol. 67, Issue 4
  • DOI: 10.1107/S0907444911001314

Conformation-independent structural comparison of macromolecules with ProSMART
journal, August 2014

  • Nicholls, Robert A.; Fischer, Marcus; McNicholas, Stuart
  • Acta Crystallographica Section D Biological Crystallography, Vol. 70, Issue 9
  • DOI: 10.1107/S1399004714016241

Crystal Structures of Δ1-Pyrroline-5-carboxylate Reductase from Human Pathogens Neisseria meningitides and Streptococcus pyogenes
journal, November 2005


UCSF Chimera?A visualization system for exploratory research and analysis
journal, January 2004

  • Pettersen, Eric F.; Goddard, Thomas D.; Huang, Conrad C.
  • Journal of Computational Chemistry, Vol. 25, Issue 13
  • DOI: 10.1002/jcc.20084

Pyrroline-5-Carboxylate Reductase Is in Pea ( Pisum sativum L.) Leaf Chloroplasts
journal, October 1989

  • Rayapati, P. John; Stewart, Cecil R.; Hack, Ethan
  • Plant Physiology, Vol. 91, Issue 2
  • DOI: 10.1104/pp.91.2.581

The Structural Biology Center 19ID undulator beamline: facility specifications and protein crystallographic results
journal, December 2005

  • Rosenbaum, Gerd; Alkire, Randy W.; Evans, Gwyndaf
  • Journal of Synchrotron Radiation, Vol. 13, Issue 1
  • DOI: 10.1107/S0909049505036721

Proline Accumulates in Plants Exposed to uv Radiation and Protects Them against uv-Induced Peroxidation
journal, April 1995

  • Saradhi, P. P.; AliaArora, S.; Prasad, K. V. S. K.
  • Biochemical and Biophysical Research Communications, Vol. 209, Issue 1
  • DOI: 10.1006/bbrc.1995.1461

Heavy metal-induced accumulation of free proline in a metal-tolerant and a nontolerant ecotype of Silene vulgaris
journal, November 1997


Pyrroline-5-carboxylate synthase activity in mammalian cells.
journal, September 1980

  • Smith, R. J.; Downing, S. J.; Phang, J. M.
  • Proceedings of the National Academy of Sciences, Vol. 77, Issue 9
  • DOI: 10.1073/pnas.77.9.5221

Proline: a multifunctional amino acid
journal, February 2010


Duplicated P5CS genes of Arabidopsis play distinct roles in stress regulation and developmental control of proline biosynthesis
journal, January 2008


Subcellular Location of Δ 1 -Pyrroline-5-Carboxylate Reductase in Root/Nodule and Leaf of Soybean
journal, August 1992

  • Szoke, Annamaria; Miao, Guo-Hua; Hong, Zonglie
  • Plant Physiology, Vol. 99, Issue 4
  • DOI: 10.1104/pp.99.4.1642

Pleiotropic Modulation of Carbon and Nitrogen Metabolism in Arabidopsis Plants Overexpressing the NAD kinase2 Gene
journal, July 2009

  • Takahashi, Hideyuki; Takahara, Kentaro; Hashida, Shin-nosuke
  • Plant Physiology, Vol. 151, Issue 1
  • DOI: 10.1104/pp.109.140665

Proline accumulation inside symbiosomes of faba bean nodules under salt stress
journal, September 1998


Transgenic Medicago truncatula plants that accumulate proline display nitrogen-fixing activity with enhanced tolerance to osmotic stress
journal, October 2006


Proline Metabolism and Its Implications for Plant-Environment Interaction
journal, January 2010

  • Verslues, Paul E.; Sharma, Sandeep
  • The Arabidopsis Book, Vol. 8
  • DOI: 10.1199/tab.0140

Improved chemical synthesis and enzymatic assay ofΔ1-pyrroline-5-car☐ylic acid
journal, March 1975


Use of TLS parameters to model anisotropic displacements in macromolecular refinement
journal, January 2001

  • Winn, M. D.; Isupov, M. N.; Murshudov, G. N.
  • Acta Crystallographica Section D Biological Crystallography, Vol. 57, Issue 1
  • DOI: 10.1107/S0907444900014736

Physiological implications of arginine metabolism in plants
journal, July 2015

  • Winter, Gudrun; Todd, Christopher D.; Trovato, Maurizio
  • Frontiers in Plant Science, Vol. 6
  • DOI: 10.3389/fpls.2015.00534

Hydrogen peroxide-induced proline and metabolic pathway of its accumulation in maize seedlings
journal, October 2009


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    • DOI: 10.1590/1678-992x-2018-0095

    Lysine metabolism and amino acid profile in maize grains from plants subjected to cadmium exposure
    journal, January 2020

    • Kato, Fabiana Hibary; Carvalho, Marcia Eugenia Amaral; Gaziola, Salete Aparecida
    • Scientia Agricola, Vol. 77, Issue 1
    • DOI: 10.1590/1678-992x-2018-0095