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Title: Functional properties and structural characterization of rice δ1-pyrroline-5-carboxylate reductase

Abstract

The majority of plant species accumulate high intracellular levels of proline to cope with hyperosmotic stress conditions. Proline synthesis from glutamate is tightly regulated at both the transcriptional and the translational levels, yet little is known about the mechanisms for post-translational regulation of the enzymatic activities involved. The gene coding in rice (Oryza sativa L.) for δ1-pyrroline-5-carboxylate (P5C) reductase, the enzyme that catalyzes the second and final step in this pathway, was isolated and expressed in Escherichia coli. The structural and functional properties of the affinity-purified protein were characterized. As for most species, rice P5C reductase was able to use in vitro either NADH or NADPH as the electron donor. However, strikingly different effects of cations and anions were found depending on the pyridine nucleotide used, namely inhibition of NADH-dependent activity and stimulation of NADPH-dependent activity. Moreover, physiological concentrations of proline and NADP+ were strongly inhibitory for the NADH-dependent reaction, whereas the NADPH-dependent activity was mildly affected. Our results suggest that only NADPH may be used in vivo and that stress-dependent variations in ion homeostasis and NADPH/NADP+ ratio could modulate enzyme activity, being functional in promoting proline accumulation and potentially also adjusting NADPH consumption during the defense against hyperosmotic stress.more » The apparent molecular weight of the native protein observed in size exclusion chromatography indicated a high oligomerization state. We also report the first crystal structure of a plant P5C reductase at 3.40-Å resolution, showing a decameric quaternary assembly. It was possible to identify dynamic structural differences among rice, human, and bacterial enzymes.« less

Authors:
 [1];  [2];  [1];  [3];  [4];  [5]
  1. University of Ferrara, Ferrara (Italy). Laboratory of Plant Physiology and Biochemistry.
  2. University of Ferrara, Ferrara (Italy). Laboratory of Plant Physiology and Biochemistry; Univ. of Konstanz, Konstanz, (Germany). Plant Physiology and Biochemistry.
  3. Univ. of Konstanz, Konstanz (Germany). Plant Physiology and Biochemistry.
  4. Argonne National Lab., Argonne, IL (United States). Synchrotron Radiation Research Section.
  5. Argonne National Lab., Argonne, IL (United States). Biosciences Div.
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES)
OSTI Identifier:
1221834
Grant/Contract Number:  
AC02-06CH11357
Resource Type:
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; proline synthesis; P5C reductase; enzyme properties; substrate ambiguity; cation and anion effects; product inhibition; oligomeric structure

Citation Formats

Forlani, Giuseppe, Bertazzini, Michele, Zarattini, Marco, Funck, Dietmar, Ruszkowski, Milosz, and Nocek, Bogusław. Functional properties and structural characterization of rice δ1-pyrroline-5-carboxylate reductase. United States: N. p., 2015. Web. doi:10.3389/fpls.2015.00565.
Forlani, Giuseppe, Bertazzini, Michele, Zarattini, Marco, Funck, Dietmar, Ruszkowski, Milosz, & Nocek, Bogusław. Functional properties and structural characterization of rice δ1-pyrroline-5-carboxylate reductase. United States. https://doi.org/10.3389/fpls.2015.00565
Forlani, Giuseppe, Bertazzini, Michele, Zarattini, Marco, Funck, Dietmar, Ruszkowski, Milosz, and Nocek, Bogusław. Tue . "Functional properties and structural characterization of rice δ1-pyrroline-5-carboxylate reductase". United States. https://doi.org/10.3389/fpls.2015.00565. https://www.osti.gov/servlets/purl/1221834.
@article{osti_1221834,
title = {Functional properties and structural characterization of rice δ1-pyrroline-5-carboxylate reductase},
author = {Forlani, Giuseppe and Bertazzini, Michele and Zarattini, Marco and Funck, Dietmar and Ruszkowski, Milosz and Nocek, Bogusław},
abstractNote = {The majority of plant species accumulate high intracellular levels of proline to cope with hyperosmotic stress conditions. Proline synthesis from glutamate is tightly regulated at both the transcriptional and the translational levels, yet little is known about the mechanisms for post-translational regulation of the enzymatic activities involved. The gene coding in rice (Oryza sativa L.) for δ1-pyrroline-5-carboxylate (P5C) reductase, the enzyme that catalyzes the second and final step in this pathway, was isolated and expressed in Escherichia coli. The structural and functional properties of the affinity-purified protein were characterized. As for most species, rice P5C reductase was able to use in vitro either NADH or NADPH as the electron donor. However, strikingly different effects of cations and anions were found depending on the pyridine nucleotide used, namely inhibition of NADH-dependent activity and stimulation of NADPH-dependent activity. Moreover, physiological concentrations of proline and NADP+ were strongly inhibitory for the NADH-dependent reaction, whereas the NADPH-dependent activity was mildly affected. Our results suggest that only NADPH may be used in vivo and that stress-dependent variations in ion homeostasis and NADPH/NADP+ ratio could modulate enzyme activity, being functional in promoting proline accumulation and potentially also adjusting NADPH consumption during the defense against hyperosmotic stress. The apparent molecular weight of the native protein observed in size exclusion chromatography indicated a high oligomerization state. We also report the first crystal structure of a plant P5C reductase at 3.40-Å resolution, showing a decameric quaternary assembly. It was possible to identify dynamic structural differences among rice, human, and bacterial enzymes.},
doi = {10.3389/fpls.2015.00565},
journal = {Frontiers in Plant Science},
number = ,
volume = 6,
place = {United States},
year = {Tue Jul 28 00:00:00 EDT 2015},
month = {Tue Jul 28 00:00:00 EDT 2015}
}

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Drought-mitigating Pseudomonas putida GAP-P45 modulates proline turnover and oxidative status in Arabidopsis thaliana under water stress
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