title: γ-Tocopherol methyltransferase from the green alga Chlamydomonas reinhardtii: functional characterization and expression analysis.
abstract: γ-Tocopherol methyltransferase (γ-TMT) (EC 2.1.1.95) is a very important enzyme in tocopherol biosynthesis in all photosynthetic organisms. In this paper, we present the functional characterization and expression analysis of γ-TMT from the unicellular green alga Chlamydomonas reinhardtii. Recombinant TMT1 enzyme was purified and characterized. The size of TMT1 subunit was estimated as 37 kDa by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE), in accordance with the predicted molecular size after TMT1 cDNA sequence. Recombinant TMT1 also showed an apparent molecular mass of 37 kDa in its native conformation, suggesting that native TMT1 has a monomeric structure similar to the plant TMTs already characterized. pH and temperature dependence of TMT1 activity were also similar to plant TMTs. Substrate specificity studies showed that Chlamydomonas TMT1 is responsible for the conversion of γ- and δ-tocopherol to α- and β-tocopherol, respectively. The kinetic properties of Chlamydomonas recombinant γ-TMT activity were studied and γ-TMT1 has a similar affinity for γ- and δ-tocopherol. Promoter sequence analysis and expression analysis by northern blot revealed that tmt1 expression is strongly upregulated by high light and downregulated by low temperature. This regulatory pattern of tmt1 expression supports the idea that γ- and α-tocopherol play specific roles in the adaptation to growth under low temperature and high light stress conditions.
authors: Departamento de Botánica, Ecología y Fisiología Vegetal, Instituto Andaluz de Biotecnología, Campus de Rabanales, Edif. C-4, 3a Planta, Universidad de Córdoba, 14071 Córdoba, Spain.
authors: Gregorio
authors: G
authors: Gálvez-Valdivieso
authors: Rosa
authors: R
authors: Cardeñosa
authors: José Manuel
authors: JM
authors: Vera
authors: Manuel
authors: M
authors: Pineda
authors: Miguel
authors: M
authors: Aguilar
mesh_terms: D001483:Base Sequence; D016825:Chlamydomonas reinhardtii / Q000201:enzymology* / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D018506:Gene Expression Regulation, Plant; D006863:Hydrogen-Ion Concentration; D008780:Methyltransferases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D018749:RNA, Plant / Q000235:genetics; D017422:Sequence Analysis, DNA; D013696:Temperature; D024505:Tocopherols / Q000378:metabolism
chemical_list: D010940:Plant Proteins; D018749:RNA, Plant; D008780:Methyltransferases; C047259:gamma-tocopherol methyltransferase; D024505:Tocopherols