Thermostable farnesyl diphosphate synthase of Bacillus stearothermophilus: crystallization and site-directed mutagenesis.
The gene for thermostable farnesyl diphosphate synthase from Bacillus stearothermophilus was cloned, sequenced, and overexpressed in Escherichia coli. The synthase was purified to homogeneity and crystallized. The enzyme carried only two cysteine residues in contrast to its counterparts from other sources, which have four to six cysteine residues. Either or both of the cysteine residues can be replaced with serine without causing a loss of the catalytic activity. The conserved arginine residue that occupies the third position from the C-terminus was also replaced with valine without significant loss of activity, but the valine mutant showed a weakened affinity for isopentenyl diphosphate.
Institute for Chemical Reaction Science, Tohoku University, Sendai, Japan.
T
T
Koyama
S
S
Obata
M
M
Osabe
K
K
Saito
A
A
Takeshita
T
T
Nishino
K
K
Ogura
D000595:Amino Acid Sequence; D001120:Arginine; D001483:Base Sequence; D003001:Cloning, Molecular; D017124:Conserved Sequence; D003460:Crystallization; D004122:Dimethylallyltranstransferase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D005658:Fungi / Q000201:enzymology; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D006358:Hot Temperature; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D013816:Thermodynamics
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D001120:Arginine; D004122:Dimethylallyltranstransferase