Abstract
Few land plants can synthesize and accumulate the osmoprotectant glycine betaine (GB) even though this metabolic trait has major adaptive importance given the prevalence of drought, hypersaline soils or cold. GB is synthesized from choline in two reactions catalyzed by choline monooxygenases (CMOs) and enzymes of the family 10 of aldehyde dehydrogenases (ALDH10s) that gained betaine aldehyde dehydrogenase activity (BADH). Homolog genes encoding CMO and ALDH10 enzymes are present in all known land plant genomes, but since GB-non-accumulators plants lack the BADH-type ALDH10 isozyme, they would be expected to also lack the CMO activity to avoid accumulation of the toxic betaine aldehyde. To explore CMOs substrate specificity, we performed amino acid sequence alignments, phylogenetic analysis, homology modeling and docking simulations. We found that plant CMOs form a monophyletic subfamily within the Rieske/mononuclear non-heme oxygenases family with two clades: CMO1 and CMO2, the latter diverging from CMO1 after gene duplication. CMO1 enzymes are present in all plants; CMO2s only in the Amaranthaceae high-GB-accumulators plants. CMO2s, and particularly their mononuclear non-heme iron domain where the active site is located, evolved at a faster rate than CMO1s, which suggests positive selection. The homology model and docking simulations of the spinach CMO2 enzyme showed at the active site three aromatic residues forming a box with which the trimethylammonium group of choline could interact through cation-π interactions, and a glutamate, which also may interact with the trimethylammonium group through a charge-charge interaction. The aromatic box and the carboxylate have been shown to be critical for choline binding in other proteins. Interestingly, these residues are conserved in CMO2 proteins but not in CMO1 proteins, where two of these aromatic residues are leucine and the glutamate is asparagine. These findings reinforce our proposal that the CMO1s physiological substrate is not choline but a still unknown metabolite.
Highlights
Land plants are sessile organisms that have evolved a great variety of strategies to escape from, or contend with, the many kinds of abiotic and biotic stresses to which they may be exposed during their lives
According to the Conserved Domain Database (CDD), the N-terminal Rieske domain of choline monooxygenases (CMOs) proteins belongs to the cd03541 (Rieske_Ro_Alpha_N_CMO) subfamily of the Rieske_Ro_Alpha_N protein family, and their C-terminal catalytic domain belongs to the cd08883 (Rho_Alpha_C_CMO-like) subfamily of the Rho_Alpha_C protein family
The current experimental evidence obtained with wild-type and transgenic plants suggests that the acquisition of the ability to synthesize Glycine betaine (GB) by evolving a betaine aldehyde dehydrogenase activity (BADH) activity should have been accompanied by other adaptations, such as the gain of a significant CMO activity
Summary
Land plants are sessile organisms that have evolved a great variety of strategies to escape from, or contend with, the many kinds of abiotic and biotic stresses to which they may be exposed during their lives. Given the observed health benefits of the intake of GB in humans [6] and animals [7], the ability of an edible plant to accumulate GB is important from an agricultural and from a nutritional point of view. Because of these reasons and because many important crops and forage plants are GB-nonaccumulators, the engineering of the ability to synthesize this osmoprotectant has been, and still is, a biotechnological goal [8]. This outcome emphasizes the need to get a deeper understanding of the enzymes involved in GB biosynthesis
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