title: Molecular Evolution of the Substrate Specificity of Chloroplastic Aldolases/Rubisco Lysine Methyltransferases in Plants.
abstract: Rubisco and fructose-1,6-bisphosphate aldolases (FBAs) are involved in CO2 fixation in chloroplasts. Both enzymes are trimethylated at a specific lysine residue by the chloroplastic protein methyltransferase LSMT. Genes coding LSMT are present in all plant genomes but the methylation status of the substrates varies in a species-specific manner. For example, chloroplastic FBAs are naturally trimethylated in both Pisum sativum and Arabidopsis thaliana, whereas the Rubisco large subunit is trimethylated only in the former species. The in vivo methylation status of aldolases and Rubisco matches the catalytic properties of AtLSMT and PsLSMT, which are able to trimethylate FBAs or FBAs and Rubisco, respectively. Here, we created chimera and site-directed mutants of monofunctional AtLSMT and bifunctional PsLSMT to identify the molecular determinants responsible for substrate specificity. Our results indicate that the His-Ala/Pro-Trp triad located in the central part of LSMT enzymes is the key motif to confer the capacity to trimethylate Rubisco. Two of the critical residues are located on a surface loop outside the methyltransferase catalytic site. We observed a strict correlation between the presence of the triad motif and the in vivo methylation status of Rubisco. The distribution of the motif into a phylogenetic tree further suggests that the ancestral function of LSMT was FBA trimethylation. In a recent event during higher plant evolution, this function evolved in ancestors of Fabaceae, Cucurbitaceae, and Rosaceae to include Rubisco as an additional substrate to the archetypal enzyme. Our study provides insight into mechanisms by which SET-domain protein methyltransferases evolve new substrate specificity.
authors.affiliation: Université Grenoble Alpes, Laboratoire de Physiologie Cellulaire & Végétale, 38041 Grenoble, France; CNRS, UMR5168, 38054 Grenoble, France; CEA, iRTSV, Laboratoire de Physiologie Cellulaire & Végétale, 38054 Grenoble, France; INRA, USC1359, 38054 Grenoble, France.
authors.forename: Sheng
authors.initials: S
authors.lastname: Ma
authors.affiliation: Université Grenoble Alpes, Laboratoire de Physiologie Cellulaire & Végétale, 38041 Grenoble, France; CNRS, UMR5168, 38054 Grenoble, France; CEA, iRTSV, Laboratoire de Physiologie Cellulaire & Végétale, 38054 Grenoble, France; INRA, USC1359, 38054 Grenoble, France.
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Martin-Laffon
authors.affiliation: Université Grenoble Alpes, Laboratoire de Physiologie Cellulaire & Végétale, 38041 Grenoble, France; CNRS, UMR5168, 38054 Grenoble, France; CEA, iRTSV, Laboratoire de Physiologie Cellulaire & Végétale, 38054 Grenoble, France; INRA, USC1359, 38054 Grenoble, France.
authors.forename: Morgane
authors.initials: M
authors.lastname: Mininno
authors.affiliation: Université Grenoble Alpes, Laboratoire de Physiologie Cellulaire & Végétale, 38041 Grenoble, France; CNRS, UMR5168, 38054 Grenoble, France; CEA, iRTSV, Laboratoire de Physiologie Cellulaire & Végétale, 38054 Grenoble, France; INRA, USC1359, 38054 Grenoble, France.
authors.forename: Océane
authors.initials: O
authors.lastname: Gigarel
authors.affiliation: Université Grenoble Alpes, 38041 Grenoble, France; CEA, iRTSV, Biologie à Grande Echelle, 38054 Grenoble, France; INSERM, U1038, 38054 Grenoble, France.
authors.forename: Sabine
authors.initials: S
authors.lastname: Brugière
authors.affiliation: Université Grenoble Alpes, Laboratoire de Physiologie Cellulaire & Végétale, 38041 Grenoble, France; CNRS, UMR5168, 38054 Grenoble, France; CEA, iRTSV, Laboratoire de Physiologie Cellulaire & Végétale, 38054 Grenoble, France; INRA, USC1359, 38054 Grenoble, France.
authors.forename: Olivier
authors.initials: O
authors.lastname: Bastien
authors.affiliation: Université Grenoble Alpes, 38041 Grenoble, France; CEA, iRTSV, Biologie à Grande Echelle, 38054 Grenoble, France; INSERM, U1038, 38054 Grenoble, France.
authors.forename: Marianne
authors.initials: M
authors.lastname: Tardif
authors.affiliation: Université Grenoble Alpes, Laboratoire de Physiologie Cellulaire & Végétale, 38041 Grenoble, France; CNRS, UMR5168, 38054 Grenoble, France; CEA, iRTSV, Laboratoire de Physiologie Cellulaire & Végétale, 38054 Grenoble, France; INRA, USC1359, 38054 Grenoble, France.
authors.forename: Stéphane
authors.initials: S
authors.lastname: Ravanel
authors.affiliation: Université Grenoble Alpes, Laboratoire de Physiologie Cellulaire & Végétale, 38041 Grenoble, France; CNRS, UMR5168, 38054 Grenoble, France; CEA, iRTSV, Laboratoire de Physiologie Cellulaire & Végétale, 38054 Grenoble, France; INRA, USC1359, 38054 Grenoble, France. Electronic address: claude.alban@cea.fr.
authors.forename: Claude
authors.initials: C
authors.lastname: Alban
mesh_terms: D000446:Aldehyde-Lyases / Q000378:metabolism*; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D002736:Chloroplasts / Q000201:enzymology*; D019143:Evolution, Molecular; D008745:Methylation; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D000072417:Protein Domains; D012273:Ribulose-Bisphosphate Carboxylase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D012273:Ribulose-Bisphosphate Carboxylase; D000446:Aldehyde-Lyases
keywords: SET-domain protein methyltransferase; chimeric enzyme; green lineage; methylation; molecular evolution; site-directed mutagenesis