Cloning and expression of 1-deoxy-d-xylulose 5-phosphate synthase cDNA from Croton stellatopilosus and expression of 2C-methyl-d-erythritol 4-phosphate synthase and geranylgeranyl diphosphate synthase, key enzymes of plaunotol biosynthesis.
1-Deoxy-d-xylulose 5-phosphate synthase (DXS, EC: 4.1.3.37), the first enzyme in the 2C-methyl-d-erythritol 4-phosphate (MEP) pathway, is known to be responsible for the rate-limiting step of isoprenoid biosynthesis in Escherichia coli and Arabidopsis thaliana. In this study, the dxs gene from Croton stellatopilosus, designated csdxs, was cloned from leaf tissue using the rapid amplification of cDNA ends (RACE) technique. Leaves of C. stellatopilosus contain plaunotol, an acyclic diterpene alcohol. The csdxs cDNA containing the open reading frame of 2163 base pairs appeared to encode a polypeptide of 720 amino acids. Analysis of the deduced amino acid sequence revealed that the NH(2)-terminus of CSDXS carried a chloroplast transit peptide, a thiamine diphosphate binding site, and a transketolase motif, which are the important characteristics of DXS enzymes in higher plants. Multiple alignments of CSDXS with other plant DXSs have indicated that CSDXS has identity ranging between 68% and 89%. Expression levels of csdxs and genes encoding key enzymes in the plaunotol biosynthetic pathway, namely 2C-methyl-d-erythritol 4-phosphate synthase (meps) and geranylgeranyl diphosphate synthase (ggpps), were analysed by measuring transcript levels in leaves of different developmental stages. The results showed that dxs, meps, and ggpps are all active in young leaves prior to full expansion when plaunotol is synthesised from the DXP precursor in chloroplasts. The dense presence of chloroplasts and oil globules in the palisade cells of these leaves support the view that these genes are involved in plaunotol biosynthesis in chloroplast-containing tissues.
Sitthithaworn
Worapan
W
Department of Pharmaceutical Chemistry and Pharmacognosy, Faculty of Pharmacy, Srinakharinwirot University, Nakonnayok 26120, Thailand.
Wungsintaweekul
Juraithip
J
Sirisuntipong
Tanawan
T
Charoonratana
Tossaton
T
Ebizuka
Yutaka
Y
De-Eknamkul
Wanchai
W
D019747:Aldose-Ketose Isomerases / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D029802:Croton / Q000201:enzymology* / Q000235:genetics* / Q000648:ultrastructure; D018076:DNA, Complementary / Q000235:genetics*; D004224:Diterpenes; D051231:Farnesyltranstransferase / Q000235:genetics / Q000378:metabolism*; D005233:Fatty Alcohols / Q000737:chemistry / Q000378:metabolism*; D020869:Gene Expression Profiling; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics / Q000378:metabolism*; D009928:Organ Specificity; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D010802:Phylogeny; D018515:Plant Leaves / Q000648:ultrastructure; D018520:Plant Shoots / Q000648:ultrastructure; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D014166:Transferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D018076:DNA, Complementary; D004224:Diterpenes; D005233:Fatty Alcohols; D009097:Multienzyme Complexes; D012333:RNA, Messenger; D010088:Oxidoreductases; C114231:1-deoxy-D-xylulose 5-phosphate reductoisomerase; D014166:Transferases; C109461:deoxyxylulose-5-phosphate synthase; D051231:Farnesyltranstransferase; D019747:Aldose-Ketose Isomerases; C047686:plaunotol