Alpha-amylase from the hyperthermophilic archaebacterium Pyrococcus furiosus. Cloning and sequencing of the gene and expression in Escherichia coli.
A gene encoding a highly thermostable alpha-amylase from the hyperthermophilic archaebacterium Pyrococcus furiosus was cloned and expressed in Escherichia coli. The nucleotide sequence of the gene predicts a 649-amino acid protein with a calculated molecular mass of 76.3 kDa, which corresponds well with the value obtained from purified enzyme using denaturing polyacrylamide gel electrophoresis. The NH2 terminus of the deduced amino acid sequence corresponds precisely to that obtained from the purified enzyme, excluding the NH2-terminal methionine. The amylase expressed in E. coli exhibits temperature-dependent activation characteristic of of the original enzyme from P. furiosus, but has a higher apparent molecular weight which is attributed to the improper formation of the native quaternary structure. No homology was found with previously characterized promotor or termination sequences. The deduced amino acid sequence displayed strong homology to the alpha-amylase A of Dictyoglomus thermophilum, an obligately anaerobic, extremely thermophilic bacterium. Evolutionary implications of this homology are discussed.
Laderman
K A
KA
Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218.
Asada
K
K
Uemori
T
T
Mukai
H
H
Taguchi
Y
Y
Kato
I
I
Anfinsen
C B
CB
D000595:Amino Acid Sequence; D001105:Archaea / Q000201:enzymology*; D001483:Base Sequence; D003001:Cloning, Molecular; D003198:Computer Simulation; D004269:DNA, Bacterial; D004789:Enzyme Activation; D004926:Escherichia coli; D008969:Molecular Sequence Data; D010957:Plasmids; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015183:Restriction Mapping; D000516:alpha-Amylases / Q000235:genetics* / Q000378:metabolism
D004269:DNA, Bacterial; D011994:Recombinant Proteins; D000516:alpha-Amylases