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Functional characterization and expression analysis of rice δ(1)-pyrroline-5-carboxylate dehydrogenase provide new insight into the regulation of proline and arginine catabolism.

Forlani G, Bertazzini M, Zarattini M, Funck D - Front Plant Sci (2015)

Bottom Line: Cations were found to modulate enzyme activity, whereas anion effects were negligible.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Science and Biotechnology, University of Ferrara Ferrara, Italy.

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.

No MeSH data available.


Kinetic features of rice P5C dehydrogenase. The activity of purified recombinant rice P5C dehydrogenase (0.5 μg) was measured for up to 30 min at 35°C in the presence of increasing concentrations of NAD(P)+(A) or L-P5C (B). Invariable substrates were fixed at the same levels as in the standard assay. Plotting of data from Michaelis–Menten graphs into the Lineweaver–Burk double reciprocal plots allowed the calculation of affinity constants and Vmax values for the NAD+- and the NADP+-dependent reaction (Table 2). The pH-dependence of enzyme activity was determined using NAD+ as the electron acceptor (C). Better fiting of the experimental results was obtained assuming the sum of two gaussian distributions (green line r2 = 0.9834) than a single gaussian (brown line; r2 = 0.9504). The 95%-confidence intervals of the adopted interpolating curve are indicated by a dotted line. The effect of Mg2+ ions in the range from 1 to 10 mM was evaluated by adding MgCl2 to the standard reaction mixture (D). In all cases, at least three replicates were carried out for each treatment, and mean values ± SE are presented.
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Figure 7: Kinetic features of rice P5C dehydrogenase. The activity of purified recombinant rice P5C dehydrogenase (0.5 μg) was measured for up to 30 min at 35°C in the presence of increasing concentrations of NAD(P)+(A) or L-P5C (B). Invariable substrates were fixed at the same levels as in the standard assay. Plotting of data from Michaelis–Menten graphs into the Lineweaver–Burk double reciprocal plots allowed the calculation of affinity constants and Vmax values for the NAD+- and the NADP+-dependent reaction (Table 2). The pH-dependence of enzyme activity was determined using NAD+ as the electron acceptor (C). Better fiting of the experimental results was obtained assuming the sum of two gaussian distributions (green line r2 = 0.9834) than a single gaussian (brown line; r2 = 0.9504). The 95%-confidence intervals of the adopted interpolating curve are indicated by a dotted line. The effect of Mg2+ ions in the range from 1 to 10 mM was evaluated by adding MgCl2 to the standard reaction mixture (D). In all cases, at least three replicates were carried out for each treatment, and mean values ± SE are presented.

Mentions: The recombinant enzyme was thoroughly characterized with respect to functional and structural properties (Table 2). Interestingly, maximal activity was found at pH 6.72, an unusual feature for an enzyme that is functionally located in the mitochondrial matrix. However, the curve for the pH-dependence of the specific activity was much better fitted as a superimposition of two Gaussian distribution curves with maxima at pH 6.70 and pH 7.79 than by a curve with a single maximum (Figure 7C). An isoelectric point of 5.60 was experimentally determined, in good agreement with that calculated in silico (5.84). The inclusion of Mg2+ ions into the reaction mixture at millimolar concentrations only slightly influenced enzyme activity, with a mild stimulation when NADP+ was used as the electron acceptor, and a slight inhibition if NAD+ was the co-factor (Figure 7D).


Functional characterization and expression analysis of rice δ(1)-pyrroline-5-carboxylate dehydrogenase provide new insight into the regulation of proline and arginine catabolism.

Forlani G, Bertazzini M, Zarattini M, Funck D - Front Plant Sci (2015)

Kinetic features of rice P5C dehydrogenase. The activity of purified recombinant rice P5C dehydrogenase (0.5 μg) was measured for up to 30 min at 35°C in the presence of increasing concentrations of NAD(P)+(A) or L-P5C (B). Invariable substrates were fixed at the same levels as in the standard assay. Plotting of data from Michaelis–Menten graphs into the Lineweaver–Burk double reciprocal plots allowed the calculation of affinity constants and Vmax values for the NAD+- and the NADP+-dependent reaction (Table 2). The pH-dependence of enzyme activity was determined using NAD+ as the electron acceptor (C). Better fiting of the experimental results was obtained assuming the sum of two gaussian distributions (green line r2 = 0.9834) than a single gaussian (brown line; r2 = 0.9504). The 95%-confidence intervals of the adopted interpolating curve are indicated by a dotted line. The effect of Mg2+ ions in the range from 1 to 10 mM was evaluated by adding MgCl2 to the standard reaction mixture (D). In all cases, at least three replicates were carried out for each treatment, and mean values ± SE are presented.
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Related In: Results  -  Collection

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Show All Figures
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Figure 7: Kinetic features of rice P5C dehydrogenase. The activity of purified recombinant rice P5C dehydrogenase (0.5 μg) was measured for up to 30 min at 35°C in the presence of increasing concentrations of NAD(P)+(A) or L-P5C (B). Invariable substrates were fixed at the same levels as in the standard assay. Plotting of data from Michaelis–Menten graphs into the Lineweaver–Burk double reciprocal plots allowed the calculation of affinity constants and Vmax values for the NAD+- and the NADP+-dependent reaction (Table 2). The pH-dependence of enzyme activity was determined using NAD+ as the electron acceptor (C). Better fiting of the experimental results was obtained assuming the sum of two gaussian distributions (green line r2 = 0.9834) than a single gaussian (brown line; r2 = 0.9504). The 95%-confidence intervals of the adopted interpolating curve are indicated by a dotted line. The effect of Mg2+ ions in the range from 1 to 10 mM was evaluated by adding MgCl2 to the standard reaction mixture (D). In all cases, at least three replicates were carried out for each treatment, and mean values ± SE are presented.
Mentions: The recombinant enzyme was thoroughly characterized with respect to functional and structural properties (Table 2). Interestingly, maximal activity was found at pH 6.72, an unusual feature for an enzyme that is functionally located in the mitochondrial matrix. However, the curve for the pH-dependence of the specific activity was much better fitted as a superimposition of two Gaussian distribution curves with maxima at pH 6.70 and pH 7.79 than by a curve with a single maximum (Figure 7C). An isoelectric point of 5.60 was experimentally determined, in good agreement with that calculated in silico (5.84). The inclusion of Mg2+ ions into the reaction mixture at millimolar concentrations only slightly influenced enzyme activity, with a mild stimulation when NADP+ was used as the electron acceptor, and a slight inhibition if NAD+ was the co-factor (Figure 7D).

Bottom Line: Cations were found to modulate enzyme activity, whereas anion effects were negligible.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Science and Biotechnology, University of Ferrara Ferrara, Italy.

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.

No MeSH data available.