Pyrroline-5-carboxylate synthase and proline biosynthesis: from osmotolerance to rare metabolic disease

Abstract

Pyrroline-5-carboxylate synthase (P5CS) is a bifunctional enzyme that exhibits glutamate kinase (GK) and gamma-glutamyl phosphate reductase (GPR) activities. The enzyme is highly relevant in humans because it belongs to a combined route for the interconversion of glutamate, ornithine and proline. The deficiency of P5CS activity in humans is associated with a rare, inherited metabolic disease. It is well established that some bacteria and plants accumulate proline in response to osmotic stress. The alignment of P5CSs from different species and analysis of the solved structures of GK and GPR reveal high sequence and structural conservation. The information acquired from different mutant enzymes with increased osmotolerant properties, together with the position of the insertion found in the longer human isoform, permit the delimitation of the regulatory site of GK and P5CS and the proposal of a model of P5CS architecture. Additionally, the GK moiety of the human enzyme has been modeled and the known clinical mutations and polymorphisms have been mapped.

Figure 1

General pathway of the proline metabolism in higher eukaryotes, with the regulation of P5CS and P5CR in plants and mammals.

Figure 2

Schematic representation of human P5CS and bacterial GK and GPR, showing the location of certain structural features.

Figure 3

(A) A ribbon and surface representation of a GK dimer from C. jejuni and (B) from E. coli where the mutated residues found in the different GK and P5CS enzymes affecting proline regulation are drawn in red. Arrows denote the location of the VN insertion (colored in black) found in the human long P5CS isoform. C: Superposition of the AAK domain of E. coli GK colored in blue (2J5T), C. jejuni GK (2AKO) colored in green, together with the modeled human GK moiety generated by using the Swiss Model Workspace, colored in pink. D: Superposition of the GPR enzymes from T. maritima (1O20) colored in yellow, S. cerevisiae (1VLU) colored in blue and the human GPR moiety of P5CS (2H5G) colored in green. E: Ribbon representation of the modeled structure of the human GK moiety, with the ADP molecule depicted by sticks, and the glutamate by spheres, and (F) the crystal structure of the human GPR moiety with the different domains, the catalytic cysteine and some structural features indicated. The residues affecting clinical mutations and polymorphisms are shown.