A complex array of factors regulate the activity of Arabidopsis thaliana δ1 -pyrroline-5-carboxylate synthetase isoenzymes to ensure their specific role in plant cell metabolism.

The first and committed step in proline synthesis from glutamate is catalyzed by δ1 -pyrroline-5-carboxylate synthetase (P5CS). Two P5CS genes have been found in most angiosperms, one constitutively expressed to satisfy proline demand for protein synthesis, the other stress-induced. Despite the number of papers to investigate regulation at the transcriptional level, to date, the properties of the enzymes have been subjected to limited study. The isolation of Arabidopsis thaliana P5CS isoenzymes was achieved through heterologous expression and affinity purification. The two proteins were characterized with respect to kinetic and biochemical properties. AtP5CS2 showed KM values in the micro- to millimolar range, and its activity was inhibited by NADP+ , ADP and proline, and by glutamine and arginine at high levels. Mg2+ ions were required for activity, which was further stimulated by K+ and other cations. AtP5CS1 displayed positive cooperativity with glutamate and was almost insensitive to inhibition by proline. In the presence of physiological, nonsaturating concentrations of glutamate, proline was slightly stimulatory, and glutamine strongly increased the catalytic rate. Data suggest that the activity of AtP5CS isoenzymes is differentially regulated by a complex array of factors including the concentrations of proline, glutamate, glutamine, monovalent cations and pyridine dinucleotides.

Guanidine production by plant homoarginine-6-hydroxylases.

Metabolism and biological functions of the nitrogen-rich compound guanidine have long been neglected. The discovery of four classes of guanidine-sensing riboswitches and two pathways for guanidine degradation in bacteria hint at widespread sources of unconjugated guanidine in nature. So far, only three enzymes from a narrow range of bacteria and fungi have been shown to produce guanidine, with the ethylene-forming enzyme (EFE) as the most prominent example. Here, we show that a related class of Fe2+- and 2-oxoglutarate-dependent dioxygenases (2-ODD-C23) highly conserved among plants and algae catalyze the hydroxylation of homoarginine at the C6-position. Spontaneous decay of 6-hydroxyhomoarginine yields guanidine and 2-aminoadipate-6-semialdehyde. The latter can be reduced to pipecolate by pyrroline-5-carboxylate reductase but more likely is oxidized to aminoadipate by aldehyde dehydrogenase ALDH7B in vivo. Arabidopsis has three 2-ODD-C23 isoforms, among which Din11 is unusual because it also accepted arginine as substrate, which was not the case for the other 2-ODD-C23 isoforms from Arabidopsis or other plants. In contrast to EFE, none of the three Arabidopsis enzymes produced ethylene. Guanidine contents were typically between 10 and 20 nmol*(g fresh weight)-1 in Arabidopsis but increased to 100 or 300 nmol*(g fresh weight)-1 after homoarginine feeding or treatment with Din11-inducing methyljasmonate, respectively. In 2-ODD-C23 triple mutants, the guanidine content was strongly reduced, whereas it increased in overexpression plants. We discuss the implications of the finding of widespread guanidine-producing enzymes in photosynthetic eukaryotes as a so far underestimated branch of the bio-geochemical nitrogen cycle and propose possible functions of natural guanidine production.