title: Functional characterization and expression analysis of rice δ(1)-pyrroline-5-carboxylate dehydrogenase provide new insight into the regulation of proline and arginine catabolism.
abstract: While intracellular proline accumulation in response to various stress conditions has been investigated in great detail, the biochemistry and physiological relevance of proline degradation in plants is much less understood. Moreover, the second and last step in proline catabolism, the oxidation of δ(1)-pyrroline-5-carboxylic acid (P5C) to glutamate, is shared with arginine catabolism. Little information is available to date concerning the regulatory mechanisms coordinating these two pathways. Expression of the gene coding for P5C dehydrogenase was analyzed in rice by real-time PCR either following the exogenous supply of amino acids of the glutamate family, or under hyperosmotic stress conditions. The rice enzyme was heterologously expressed in E. coli, and the affinity-purified protein was thoroughly characterized with respect to structural and functional properties. A tetrameric oligomerization state was observed in size exclusion chromatography, which suggests a structure of the plant enzyme different from that shown for the bacterial P5C dehydrogenases structurally characterized to date. Kinetic analysis accounted for a preferential use of NAD(+) as the electron acceptor. Cations were found to modulate enzyme activity, whereas anion effects were negligible. Several metal ions were inhibitory in the micromolar range. Interestingly, arginine also inhibited the enzyme at higher concentrations, with a mechanism of uncompetitive type with respect to P5C. This implies that millimolar levels of arginine would increase the affinity of P5C dehydrogenase toward its specific substrate. Results are discussed in view of the involvement of the enzyme in either proline or arginine catabolism.
authors: Department of Life Science and Biotechnology, University of Ferrara Ferrara, Italy.
authors: Giuseppe
authors: G
authors: Forlani
authors: Department of Life Science and Biotechnology, University of Ferrara Ferrara, Italy ; Biology Section, Department of Plant Physiology and Biochemistry, University of Konstanz Konstanz, Germany.
authors: Michele
authors: M
authors: Bertazzini
authors: Department of Life Science and Biotechnology, University of Ferrara Ferrara, Italy.
authors: Marco
authors: M
authors: Zarattini
authors: Biology Section, Department of Plant Physiology and Biochemistry, University of Konstanz Konstanz, Germany.
authors: Dietmar
authors: D
authors: Funck
keywords: cation and anion effects; enzyme properties; gene expression; plant response to stress conditions; proline and arginine catabolism