title: Arabidopsis Mg-Protoporphyrin IX Methyltransferase Activity and Redox Regulation Depend on Conserved Cysteines.
abstract: Redox regulation is an essential post-translational regulatory mechanism in prokaryotes and eukaryotes. The reversible oxidation and reduction of cysteine residues of proteins is also important in photosynthetic organisms to control enzymatic activities, protein stability and the interaction with other proteins of chloroplast-localized proteins. Several enzymes of the plant tetrapyrrole biosynthesis pathway have been identified to be redox regulated by thioredoxins (TRXs) and NADPH-dependent thioredoxin reductase C (NTRC). Among these proteins, Mg protoporphyrin IX methyltransferase (encoded by CHLM) was identified to be activated and stabilized by interaction with NTRC. CHLM catalyzes a methyl group transfer by using S-adenosylmethionine (SAM). Here we demonstrate that three conserved cysteine residues of Arabidopsis CHLM are essential for catalytic function and redox-dependent activation of the enzyme. In vitro and in planta biochemical assays of recombinant CHLM and the Arabidopsis chlm knockout mutant overexpressing wild-type and cysteine substitution mutants of CHLM revealed modified methyltransferase activity, when the conserved cysteine residues of CHLM are replaced by serine. While C177 is responsible for redox-dependent enzyme activation, exchange of the two cysteine residues, C111 and C115, has a strong impact on enzyme activity. The modified CHLM activity of single and double mutants with cysteine substitution is presented, and the role of each cysteine residue is discussed based on a modeled structure of CHLM. These studies contribute to enhanced understanding of the physiological and enzymatic significance of redox-regulated CHLM.
authors.affiliation: Institute of Biology/Plant Physiology, Humboldt-University Berlin, D-10115 Berlin, Germany.
authors.forename: Andreas S
authors.initials: AS
authors.lastname: Richter
authors.affiliation: Institute of Biology/Plant Physiology, Humboldt-University Berlin, D-10115 Berlin, Germany.
authors.forename: Peng
authors.initials: P
authors.lastname: Wang
authors.affiliation: Institute of Biology/Plant Physiology, Humboldt-University Berlin, D-10115 Berlin, Germany bernhard.grimm@hu-berlin.de.
authors.forename: Bernhard
authors.initials: B
authors.lastname: Grimm
mesh_terms: D017360:Arabidopsis / Q000201:enzymology*; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D017124:Conserved Sequence; D003545:Cysteine / Q000378:metabolism*; D055786:Gene Knockout Techniques; D005816:Genetic Complementation Test; D008780:Methyltransferases / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship
chemical_list: D029681:Arabidopsis Proteins; D011994:Recombinant Proteins; D008780:Methyltransferases; C000612820:CHLM protein, Arabidopsis; C060234:magnesium-protoporphyrin methyltransferase; D003545:Cysteine
keywords: Chlorophyll; Methyltransferase; NTRC and thioredoxin; Post-translational modification; Redox regulation; Tetrapyrrole biosynthesis