Thermostable farnesyl diphosphate synthase of Bacillus stearothermophilus: molecular cloning, sequence determination, overproduction, and purification.
The structural gene for thermostable farnesyl diphosphate synthase from Bacillus stearothermophilus was cloned, sequenced, and overexpressed in Escherichia coli cells. A 1,260-nucleotide sequence of the cloned fragment was determined. This sequence specifies an open reading frame of 891 nucleotides for farnesyl diphosphate synthase. The deduced amino acid sequence shows a 42% similarity with that of E. coli FPP synthase [Fujisaki et al. (1990) J. Biochem. 108, 995-1000]. Comparison with prenyltransferases from a wide range of organisms, from bacteria to human, revealed the presence of seven highly conserved regions. In contrast to thermolabile prenyltransferases, which have four to six cysteine residues, the thermostable farnesyl diphosphate synthase carries only two cysteine residues. This enzyme is also unique in that some of the amino acids that are fully conserved in equivalents from other sources are replaced by functionally different amino acids. Construction of an overproducing strain provided a sufficient supply of this enzyme and it was purified to homogeneity. The purified recombinant enzyme is immunochemically identical with the native B. stearothermophilus enzyme, and it is not inactivated even after treatment at 65 degrees C for 70 min.
Koyama
T
T
Institute for Chemical Reaction Science, Tohoku University, Sendai.
Obata
S
S
Osabe
M
M
Takeshita
A
A
Yokoyama
K
K
Uchida
M
M
Nishino
T
T
Ogura
K
K
D019883:Alkyl and Aryl Transferases; D000595:Amino Acid Sequence; D001483:Base Sequence; D002855:Chromatography, Thin Layer; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D051228:Geranyltranstransferase; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D015183:Restriction Mapping; D013696:Temperature; D014166:Transferases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification
D004269:DNA, Bacterial; D011994:Recombinant Proteins; D014166:Transferases; D019883:Alkyl and Aryl Transferases; D051228:Geranyltranstransferase