Evolution of plant δ1-pyrroline-5-carboxylate reductases from phylogenetic and structural perspectives

Abstract

Proline plays a crucial role in cell growth and stress responses, and its accumulation is essential for the tolerance of adverse environmental conditions in plants. Two routes are used to biosynthesize proline in plants. The main route uses glutamate as a precursor, while in the other route proline is derived from ornithine. The terminal step of both pathways, the conversion of δ1-pyrroline-5-carboxylate (P5C) to L-proline, is catalyzed by P5C reductase (P5CR) using NADH or NADPH as a cofactor. Since P5CRs are important housekeeping enzymes, they are conserved across all domains of life and appear to be relatively unaffected throughout evolution. However, global analysis of these enzymes unveiled significant functional diversity in the preference for cofactors (NADPH vs. NADH), variation in metal dependence and the differences in the oligomeric state. In our study we investigated evolutionary patterns through phylogenetic and structural analysis of P5CR representatives from all kingdoms of life, with emphasis on the plant species. We also attempted to correlate local sequence/structure variation among the functionally and structurally characterized members of the family.

Functional Characterization of Four Putative δ1-Pyrroline-5-Carboxylate Reductases from Bacillus subtilis

“…In bacteria, the same reaction is accomplished by two enzymes, a γ-glutamyl kinase that catalyzes glutamate phosphorylation and a γ-glutamyl phosphate reductase that reduces the product to glutamate semialdehyde, which in solution spontaneously cyclizes to P5C (Chen et al, 2006 ; Csonka and Leisinger, 2007 ). In both cases, the latter is reduced to proline by an enzyme, P5C reductase (Forlani et al, 2015c ). Alternatively, ornithine may function as the precursor, being a pyridoxal phosphate-dependent ornithine δ-aminotransferase (δOAT) able to convert ornithine to P5C (Zaprasis et al, 2014 ; Winter et al, 2015 ).…”

“…A recent survey of the over 37,000 genes that by sequence homology have been annotated as putative P5C reductases in the NCBI database pointed out that most species possess only one gene or have lineage-specific duplications (Forlani et al, 2015c ). In several bacteria and archaea, however, distant and relatively fast-evolving paralogs have been found (Forlani et al, 2015c ) that may represent potential examples of subfunctionalization (Fichman et al, 2015 ). Yet, in a vast majority of cases the products of these isogenes have not been well-characterized.…”

“…Sequence identities are highlighted in red and similarities are displayed as red letters. The conserved sequence motifs are colored and labeled according to the nomenclature introduced in previous studies (Forlani et al, 2015c ; motif B in red, motif C in blue, motif D in magenta and motif E in green).…”

Phytotoxicity of aminobisphosphonates targeting bothδ1-pyrroline-5-carboxylate reductase and glutamine synthetase

“…In the presence of redundant biosynthetic routes, proline starvation cannot be achieved through inhibition of the enzyme that catalyses the rate‐limiting reaction in either pathway. Nevertheless, as the two routes share the last reaction, namely the reduction of δ ‐pyrroline‐5‐carboxylate (P5C) by a P5C reductase (EC 1.5.1.2), this result might be accomplished by specific inhibitors of the latter enzyme …”

“…Nevertheless, as the two routes share the last reaction, namely the reduction of 1 -pyrroline-5-carboxylate (P5C) by a P5C reductase (EC 1.5.1.2), this result might be accomplished by specific inhibitors of the latter enzyme. 19 In a first attempt, the ability to inhibit Arabidopsis thaliana P5C reductase was evaluated on a group of derivatives of aminomethylenebisphosphonic acid. Three compounds caused a dose-proportional reduction in the catalytic rate in the micromolar to millimolar range.…”