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 Dic tyostelium discoideum has been cloned and sequenced. The 855 bp cDNA o f this gene contains the open reading frame (ORF) encoding 232 amino a cids with a predicted molecular mass of approx. 26 kDa. Southern blot analysis indicated the presence of a single gene for GTP-CH in Dictyos telium. PCR amplification of the ORF from chromosomal DNA and sequenci ng showed the existence of a 101 bp intron in the GTP-CH gene of Dicty ostelium discoideum. The amino acid sequence has 47% and 49% positiona l 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. GT P-CH of Dictyostelium is heat-stable and showed maximal activity at 60 degrees C. The K-m value for GTP is 50 mu M.