Molecular cloning of a cDNA coding for GTP cyclohydrolase I from Dictyostelium discoideum.
The GTP cyclohydrolase I (GTP-CH) gene of the cellular slime mould Dictyostelium discoideum has been cloned and sequenced. The 855 bp cDNA of this gene contains the open reading frame (ORF) encoding 232 amino acids with a predicted molecular mass of approx. 26 kDa. Southern blot analysis indicated the presence of a single gene for GTP-CH in Dictyostelium. PCR amplification of the ORF from chromosomal DNA and sequencing showed the existence of a 101 bp intron in the GTP-CH gene of Dictyostelium discoideum. The amino acid sequence has 47% and 49% positional identity to those of the human and yeast enzymes respectively. Most of the sequence variation between species is located in the N-terminal part of the protein. The overall identity with the E. coli protein is markedly lower. The enzyme was expressed in E. coli and purified as a 68 kDa fusion protein with the maltose-binding protein of E. coli. GTP-CH of Dictyostelium is heat-stable and showed maximal activity at 60 degrees C. The Km value for GTP is 50 microM.
Witter
K
K
GSF-Institut für Klinische Molekularbiologie, München, Germany.
Cahill
D J
DJ
Werner
T
T
Ziegler
I
I
Rödl
W
W
Bacher
A
A
Gütlich
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004023:Dictyostelium / Q000201:enzymology* / Q000235:genetics; D004331:Drosophila melanogaster; D006136:GTP Cyclohydrolase / Q000235:genetics*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D051381:Rats; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D006136:GTP Cyclohydrolase