Cloning, expression, and characterization of cDNAs encoding Arabidopsis thaliana squalene synthase.
We have isolated and characterized two overlapping cDNA clones for Arabidopsis thaliana squalene synthase. Their nucleotide sequences contained an open reading frame for a 410-amino acid polypeptide (calculated molecular mass, 47 kDa). The deduced amino acid sequence of the Arabidopsis polypeptide was significantly homologous (42-44% identical) to the sequences of known squalene synthases of several species, from yeast to man, but it was much less homologous to that of tomato phytoene synthase. To express the Arabidopsis enzyme in Escherichia coli, the entire coding region was subcloned into an expression vector. A cell-free extract of E. coli transformed with the recombinant plasmid, in the presence of NADPH and Mg2+, efficiently converted [14C]farnesyl diphosphate into squalene. On the other hand, in the absence of NADPH and the presence of Mn2+, the cell-free extract formed dehydrosqualene as a secondary product. Another E. coli extract expressing mouse squalene synthase showed the same activity as the Arabidopsis enzyme. Therefore, both the structure and reaction mechanism of squalene synthases are markedly conserved in taxonomically remote eukaryotes.
Nakashima
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T
Faculty of Science, Himeji Institute of Technology, Hyogo, Japan.
Inoue
T
T
Oka
A
A
Nishino
T
T
Osumi
T
T
Hata
S
S
D019883:Alkyl and Aryl Transferases; D000595:Amino Acid Sequence; D000818:Animals; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D016384:Consensus Sequence; D017931:DNA Primers; D018076:DNA, Complementary; D004926:Escherichia coli; D013186:Farnesyl-Diphosphate Farnesyltransferase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D015870:Gene Expression; D017343:Genes, Plant; D005822:Genetic Vectors; D051232:Geranylgeranyl-Diphosphate Geranylgeranyltransferase; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010802:Phylogeny; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D014166:Transferases / Q000737:chemistry / Q000235:genetics; D015003:Yeasts / Q000235:genetics
D017931:DNA Primers; D018076:DNA, Complementary; D011994:Recombinant Proteins; D014166:Transferases; D019883:Alkyl and Aryl Transferases; D013186:Farnesyl-Diphosphate Farnesyltransferase; D051232:Geranylgeranyl-Diphosphate Geranylgeranyltransferase