Reviewer #1 (Public Review): Guanidine production by plant homoarginine-6-hydroxylases
2024
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 Fe 2+ -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. eLife assessmentThis fundamental study advances our understanding of nitrogen metabolism by identifying a new type of guanidine-forming enzyme in eukaryotes.The key claims of the article are convincingly supported by the data, with meticulous biochemical, cellular, and in vivo studies on guanidine production.The work will stimulate interest in the cellular roles of homoarginine, and, more generally, in the biochemistry and metabolism of guanidine derivatives.