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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.


Related in: MedlinePlus

Thermal stability of rice P5C dehydrogenase. The activity rate of the purified enzyme was measured for up to 5 min under standard assay conditions at increasing temperatures (A). Replotting data in the so-called Arrhenius plot (B) allowed the calculation of the activation energy (Table 2). Thermal stability of the enzyme was determined by incubating aliquots for increasing time at increasing temperature in the absence of substrates (C). After the indicated times, the aliquots were immediately re-equilibrated on ice and the residual activity was then measured at 35°C, and expressed as percentage of activity in untreated controls. Three replicates were carried out for each treatment, and means ± SE over replicates are shown.
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Figure 9: Thermal stability of rice P5C dehydrogenase. The activity rate of the purified enzyme was measured for up to 5 min under standard assay conditions at increasing temperatures (A). Replotting data in the so-called Arrhenius plot (B) allowed the calculation of the activation energy (Table 2). Thermal stability of the enzyme was determined by incubating aliquots for increasing time at increasing temperature in the absence of substrates (C). After the indicated times, the aliquots were immediately re-equilibrated on ice and the residual activity was then measured at 35°C, and expressed as percentage of activity in untreated controls. Three replicates were carried out for each treatment, and means ± SE over replicates are shown.

Mentions: When the purified P5C dehydrogenase was assayed as a function of the temperature, maximal initial activity was obtained at a temperature as low as 46°C (Figure 9A). The corresponding activation energy, calculated by the Arrhenius plot (Figure 9B), was 54.6 ± 3.7 kJ mol−1. Moreover, the prolonged incubation of the rice protein at temperatures in the 40 to 50°C range in the absence of its substrates caused a dramatic and rapid loss of activity (Figure 9C). A treatment at 47.5°C for 5 min resulted in a 50%-inactivation of the enzyme. Inactivation curves did not follow the conventional one-phase exponential decay. The best fit of data was obtained assuming a two phase-decay with a very short (fast) and a moderate (slow) half-life. At 40°C, half-life (fast) and half-life (slow) were 5.2 ± 0.9 min and 507 ± 219 min, whereas at 45°C they were 1.8 ± 0.6 min and 49 ± 4 min, respectively.


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)

Thermal stability of rice P5C dehydrogenase. The activity rate of the purified enzyme was measured for up to 5 min under standard assay conditions at increasing temperatures (A). Replotting data in the so-called Arrhenius plot (B) allowed the calculation of the activation energy (Table 2). Thermal stability of the enzyme was determined by incubating aliquots for increasing time at increasing temperature in the absence of substrates (C). After the indicated times, the aliquots were immediately re-equilibrated on ice and the residual activity was then measured at 35°C, and expressed as percentage of activity in untreated controls. Three replicates were carried out for each treatment, and means ± SE over replicates are shown.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4525382&req=5

Figure 9: Thermal stability of rice P5C dehydrogenase. The activity rate of the purified enzyme was measured for up to 5 min under standard assay conditions at increasing temperatures (A). Replotting data in the so-called Arrhenius plot (B) allowed the calculation of the activation energy (Table 2). Thermal stability of the enzyme was determined by incubating aliquots for increasing time at increasing temperature in the absence of substrates (C). After the indicated times, the aliquots were immediately re-equilibrated on ice and the residual activity was then measured at 35°C, and expressed as percentage of activity in untreated controls. Three replicates were carried out for each treatment, and means ± SE over replicates are shown.
Mentions: When the purified P5C dehydrogenase was assayed as a function of the temperature, maximal initial activity was obtained at a temperature as low as 46°C (Figure 9A). The corresponding activation energy, calculated by the Arrhenius plot (Figure 9B), was 54.6 ± 3.7 kJ mol−1. Moreover, the prolonged incubation of the rice protein at temperatures in the 40 to 50°C range in the absence of its substrates caused a dramatic and rapid loss of activity (Figure 9C). A treatment at 47.5°C for 5 min resulted in a 50%-inactivation of the enzyme. Inactivation curves did not follow the conventional one-phase exponential decay. The best fit of data was obtained assuming a two phase-decay with a very short (fast) and a moderate (slow) half-life. At 40°C, half-life (fast) and half-life (slow) were 5.2 ± 0.9 min and 507 ± 219 min, whereas at 45°C they were 1.8 ± 0.6 min and 49 ± 4 min, respectively.

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.


Related in: MedlinePlus