Removal of feedback inhibition of delta 1-pyrroline-5-carboxylate synthetase, a bifunctional enzyme catalyzing the first two steps of proline biosynthesis in plants.

delta 1-Pyrroline-5-carboxylate synthetase (P5CS) catalyzes the first two steps in proline biosynthesis in plants. The Vigna aconitifolia P5CS cDNA was expressed in Escherichia coli, and the enzyme was purified to homogeneity. The Vigna P5CS exhibited two activities, gamma-glutamyl kinase (gamma-GK) and glutamic acid-5-semialdehyde (GSA) dehydrogenase. The gamma-GK activity of the P5CS was detected by the hydroxamate assay and by a [14C]glutamate assay. The native molecular mass of the P5CS was 450 kDa with six identical subunits. The Vigna P5CS showed a Km of 3.6 mM for glutamate, while the Km for ATP was 2.7 mM. The gamma-GK activity of the P5CS was competitively inhibited by proline, while its GSA dehydrogenase activity was insensitive to proline. In addition, a protein inhibitor of the P5CS was observed in the plant cell. Western blot showed that the level of the P5CS was enhanced in Vigna root under salt stress. A single substitution of an alanine for a phenylalanine at amino acid residue 129 of the P5CS resulted in a significant reduction of proline feedback inhibition. The 50% inhibition values of gamma-GK activity of the wild type and the mutant P5CS were observed at 5 mM and 960 mM proline, respectively. The other properties of the mutant P5CS remained unchanged. These results may allow genetic manipulation of proline biosynthesis and overproduction of proline in plants for conferring water stress tolerance.