Ser238 is essential for ensuring differential effects of chloride anions and proline on the activity of Arabidopsis thaliana δ1-pyrroline-5-carboxylate reductase.
Many plant species accumulate proline as a mechanism to withstand various abiotic stress conditions. Proline synthesis proceeds from either glutamate or ornithine, but in both pathways the last step is catalysed by a δ1-pyrroline-5-carboxylate (P5C) reductase (EC 1.5.1.12) showing substrate ambiguity. However, the use of NADH or NADPH implies different turnover number and post-translational regulation: physiological levels of free proline feed-back inhibit only the NADH-dependent activity, whereas Cl- anions in the 20 to 200 mM range stimulate the catalytic rate only if NADPH is the hydride donor. To elucidate the molecular basis of such differences we focused on Ser238 of Arabidopsis thaliana P5C reductase, a highly conserved amino acid residue that is involved in P5C binding and had been shown to form a hydrogen bond with aminomethylene-bisphosphonic acids, leading to enzyme inhibition. Site-directed mutagenesis allowed to obtain a mutated form of the enzyme in which Ser238 has been replaced with Ala. The p.Ser238Ala P5C reductase has been expressed in E. coli, affinity purified and thoroughly characterized. Results showed the abolishment of most differences when using either nicotinamide adenine dinucleotide as the cosubstrate. Data represent a first step toward protein engineering aiming at modulating proline biosynthesis under stress.