Enzymology and Regulation of δ1-Pyrroline-5-Carboxylate Synthetase 2 From Rice

Abstract

Under several stress conditions, such as excess salt and drought, many plants accumulate proline inside the cell, which is believed to help counteracting the adverse effects of low water potential. This increase mainly relies upon transcriptional induction of δ1-pyrroline-5-carboxylate synthetase (P5CS), the enzyme that catalyzes the first two steps in proline biosynthesis from glutamate. P5CS mediates both the phosphorylation of glutamate and the reduction of γ-glutamylphosphate to glutamate-5-semialdehyde, which spontaneously cyclizes to δ1-pyrroline-5-carboxylate (P5C). In most higher plants, two isoforms of P5CS have been found, one constitutively expressed to satisfy proline demand for protein synthesis, the other stress-induced. Despite the number of papers to investigate the regulation of P5CS at the transcriptional level, to date, the properties of the enzyme have been only poorly studied. As a consequence, the descriptions of post-translational regulatory mechanisms have largely been limited to feedback-inhibition by proline. Here, we report cloning and heterologous expression of P5CS2 from Oryza sativa. The protein has been fully characterized from a functional point of view, using an assay method that allows following the physiological reaction of the enzyme. Kinetic analyses show that the activity is subjected to a wide array of regulatory mechanisms, ranging from product inhibition to feedback inhibition by proline and other amino acids. These findings confirm long-hypothesized influences of both, the redox status of the cell and nitrogen availability, on proline biosynthesis.

Intriguing Role of Proline in Redox Potential Conferring High Temperature Stress Tolerance

“…Through diverse molecular methods, key genes involved in proline biosynthesis and degradation (proline cycle; Figure 1 ) have been identified in plants ( Deuschle et al, 2001 ). The enzymes P5CS1 and P5CS2 (EC 2.7.2.11, two isoforms) utilize either NADH or NADPH ( Chen et al, 2006 ; Forlani et al, 2017 ) and ATP to produce P5C and their activities are subjected to feedback inhibition by proline, other amino acids and cofactors ( Sabbioni et al, 2021 ). Millimolar levels of proline have been found inhibitory to rice P5CS2 enzyme.…”

“…Millimolar levels of proline have been found inhibitory to rice P5CS2 enzyme. Similarly, analogs of proline like azetidine-2-carboxylate, pipecolate, hydroxyproline, phosphonoproline displayed the ability to bind to the enzyme rice P5CS2 and inhibit the activity aside cofactors such as NAD + and ADP ( Sabbioni et al, 2021 ). These results infer that structural analogs of proline inhibit the P5CS2 enzyme like that of proline feed-back inhibition.…”

Structural basis of dynamic P5CS filaments

“…The full-length wild-type or mutant P5CS (100 nM protein) activity was determined in the reaction buffer containing 25 mM HEPES pH 7.5, and 10 mM l -glutamate (Sigma), with added 20 mM MgCl 2 , 10 mM ATP (Takara), and 0.5 mM NADPH (Roche) used to initiate the reaction ( Magini et al, 2019 ; Sabbioni et al, 2021 ), then the reaction was monitored at 37°C in an MD-SpectraMax i3 plate reader and absorbance at 340 nm was measured every 20 s for 10 min (one experiment, n = 3). The NADPH concentration was converted from A340 with the standard curve determined at the same experiment.…”

The Emerging Role of Proline in the Establishment and Functioning of Legume-Rhizobium Symbiosis

“…A maximum likelihood tree reconstruction suggests that this legume-specific form derives from a duplication of P5CS2 ( Figure 2 ). It would be interesting to assess whether Mt P5CS3 is also subjected to peculiar post-translational regulation mechanisms, such as a lack of feed-back inhibition by proline, yet this aspect has been poorly investigated to date ( Sabbioni et al, 2021 ). Anyway, its emergence seems to imply that a higher demand for proline is connected with symbiotic nitrogen fixation, and further strengthens the possibility that high proline levels are needed for an optimal functioning of the nodule under stress.…”

“…Although the possibility that proline may act as ROS scavenger has not been confirmed ( Forlani et al, 2019 ), several recent studies showed that the interconversion of glutamate and proline may regulate the redox balance and the energy status of the cell in connection with other redox shuttles ( Zheng et al, 2021 ). Accordingly, most enzymes in proline metabolism influence, and are regulated by, the intracellular NAD(P) + /NAD(P)H ratio ( Giberti et al, 2014 ; Sabbioni et al, 2021 ). Therefore, active proline metabolism in nodules could be involved in generating ROS and/or maintaining redox levels required for a proper establishment of the symbiosis.…”