Glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic archaebacterium Pyrococcus woesei: characterization of the enzyme, cloning and sequencing of the gene, and expression in Escherichia coli.
The glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic archaebacterium Pyrococcus woesei (optimal growth temperature, 100 to 103 degrees C) was purified to homogeneity. This enzyme was strictly phosphate dependent, utilized either NAD+ or NADP+, and was insensitive to pentalenolactone like the enzyme from the methanogenic archaebacterium Methanothermus fervidus. The enzyme exhibited a considerable thermostability, with a 44-min half-life at 100 degrees C. The amino acid sequence of the glyceraldehyde-3-phosphate dehydrogenase from P. woesei was deduced from the nucleotide sequence of the coding gene. Compared with the enzyme homologs from mesophilic archaebacteria (Methanobacterium bryantii, Methanobacterium formicicum) and an extremely thermophilic archaebacterium (Methanothermus fervidus), the primary structure of the P. woesei enzyme exhibited a strikingly high proportion of aromatic amino acid residues and a low proportion of sulfur-containing residues. The coding gene of P. woesei was expressed at a high level in Escherichia coli, thus providing an ideal basis for detailed structural and functional studies of that enzyme.
Zwickl
P
P
Max-Planck-Institut für Biochemie, Martinsried, Federal Republic of Germany.
Fabry
S
S
Bogedain
C
C
Haas
A
A
Hensel
R
R
D000595:Amino Acid Sequence; D001105:Archaea / Q000201:enzymology* / Q000235:genetics; D001419:Bacteria / Q000201:enzymology*; D001483:Base Sequence; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005798:Genes, Bacterial; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006358:Hot Temperature; D008969:Molecular Sequence Data; D010957:Plasmids; D011487:Protein Conformation; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D013045:Species Specificity
D004269:DNA, Bacterial; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D004262:DNA Restriction Enzymes