Cloning and functional characterization of 2-C-methyl-D-erythritol 4-phosphate cytidyltransferase (GbMECT) gene from Ginkgo biloba.
2-C-methyl-D-erythritol 4-phosphate cytidyltransferase (MECT), the third enzyme of the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway, catalyzes formation of 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol from MEP. GbMECT, presumably involved in ginkgolide biosynthesis, was cloned and characterized from Ginkgo biloba embryonic roots. The protein containing the N-terminal chloroplast transit peptide consisted of 327 amino acid residues. Complementation of GbMECT with Escherichia coli NMW33, ygbP (EcMECT) knock-out mutant, rescued the mutant, confirming the function of the protein. Transcription levels of GbMECT remained generally constant in embryonic roots and leaves for 1 month. Full 88 N-terminal residues were necessary to deliver the protein into the chloroplast as shown by protein-targeting analysis with GFP as a reporter protein in Arabidopsis thaliana protoplasts.
Kim
Sang-Min
SM
Program in Applied Life Chemistry, School of Agricultural Biotechnology, Seoul National University, Sinlim-dong, Gwanak-gu, Seoul 151-921, Republic of Korea; Plant Metabolism Research Center, Kyung Hee University, Yongin 449-701, Republic of Korea.
Kuzuyama
Tomohisa
T
Chang
Yung-Jin
YJ
Kwon
Hyung-Jin
HJ
Kim
Soo-Un
SU
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004896:Erythritol / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D019143:Evolution, Molecular; D018506:Gene Expression Regulation, Plant; D020441:Ginkgo biloba / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development; D046934:Ginkgolides / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010770:Phosphotransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D010802:Phylogeny; D011523:Protoplasts / Q000378:metabolism; D016415:Sequence Alignment; D013403:Sugar Phosphates / Q000737:chemistry / Q000378:metabolism*; D014158:Transcription, Genetic / Q000235:genetics
C114232:2-C-methylerythritol 4-phosphate; D018076:DNA, Complementary; D046934:Ginkgolides; D013403:Sugar Phosphates; D010770:Phosphotransferases; D004896:Erythritol