Bona fide choline monoxygenases evolved in Amaranthaceae plants from oxygenases of unknown function: Evidence from phylogenetics, homology modeling and docking studies

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.

Roles of Endogenous Glycinebetaine in Plant Abiotic Stress Responses

“…Taken together, transgenesis of only one gene from a biosynthetic pathway is usually not enough to achieve the intended outcome. Limiting factors for GB biosynthesis can be the availability of choline, activity of the biosynthetic enzymes and their specificities toward the substrates, as well as the subcellular localization of the enzymes and their respective substrates (Huang et al 2000;Nuccio et al 1998Nuccio et al , 2000Kumar et al 2004;Muñoz-Clares et al 2014;Carrillo-Campos et al 2018). Modifications to the single-gene overexpression approaches are represented by gene stacking, a transgenesis method in which combinations of constructs harbor more than one gene and can be transferred under one selection (Zorrilla-López et al 2013).…”

“…Significant sequence-specific differences have been discovered in the GB biosynthesis isoenzymes. Phylogenetic studies show that all land plant species have genes encoding for CMO enzymes (Carrillo-Campos et al 2018). The CMO genes are present in two clades, CMO1 and CMO2, whereby CMO2 has diverged from the CMO1 after genome duplication.…”

Osmoprotectant-Mediated Abiotic Stress Tolerance in Plants

“…Taken together, transgenesis of only one gene from a biosynthetic pathway is usually not enough to achieve the intended outcome. Limiting factors for GB biosynthesis can be the availability of choline, activity of the biosynthetic enzymes and their specificities toward the substrates, as well as the subcellular localization of the enzymes and their respective substrates (Huang et al 2000;Nuccio et al 1998Nuccio et al , 2000Kumar et al 2004;Muñoz-Clares et al 2014;Carrillo-Campos et al 2018). Modifications to the single-gene overexpression approaches are represented by gene stacking, a transgenesis method in which combinations of constructs harbor more than one gene and can be transferred under one selection (Zorrilla-López et al 2013).…”

“…Significant sequence-specific differences have been discovered in the GB biosynthesis isoenzymes. Phylogenetic studies show that all land plant species have genes encoding for CMO enzymes (Carrillo-Campos et al 2018). The CMO genes are present in two clades, CMO1 and CMO2, whereby CMO2 has diverged from the CMO1 after genome duplication.…”