Molecular analysis of the essential gene for adenylate kinase from the fission yeast Schizosaccharomyces pombe.
The enzyme-catalyzed transfer of the terminal phosphoryl group from ATP to an acceptor molecule is an important reaction in a wide variety of biological processes. I demonstrate here the essential function of an ATP:AMP phosphotransferase (adenylate kinase) in the fission yeast Schizosaccharomyces pombe. A cDNA clone encoding immunoreactive adenylate kinase from S. pombe was isolated from a lambda gt11 expression library by cross-reaction with antibodies raised against the recently characterized ADK1 enzyme from the budding yeast Saccharomyces cerevisiae. Subsequent cloning and nucleotide sequence analysis of the S. pombe adenylate kinase gene, adk1, revealed a coding region of 660 nucleotides. The alignment of the two amino acid sequences from S. cerevisiae and S. pombe shows 67% identity. By gene disruption and tetrad analysis it is demonstrated that adk1 is absolutely essential for cell viability. This is in contrast to the ADK1 gene of S. cerevisiae, the deletion of which was shown to lead to a slower cell growth rate rather than to a lethal phenotype. Expression of adk1 in the S. cerevisiae ADK1 deletion strain restored normal cell growth, demonstrating that ADK1 and adk1 are functionally interchangeable. However, despite lack of absolute substrate specificity of adenylate kinases, adk1 could not complement the loss of function of the guanylate kinase encoding gene in a S. cerevisiae null mutant strain, thus highlighting the functional uniqueness of each nucleoside monophosphate kinase. Using suitable expression vectors, large amounts of active adk1 enzyme were produced in either yeast species and in E. coli. The purified enzyme exhibits a high preference for adenine nucleotides, with ATP being a 10 times more efficient phosphoryl donor than GTP.
Konrad
M
M
Max-Planck-Institute for Biophysical Chemistry, Department of Molecular Genetics, Göttingen, Germany.
D000263:Adenylate Kinase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D015825:Chromosomes, Fungal; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000235:genetics / Q000302:isolation & purification; D004274:DNA, Recombinant / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005800:Genes, Fungal; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D012568:Schizosaccharomyces / Q000201:enzymology* / Q000235:genetics*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D004271:DNA, Fungal; D004274:DNA, Recombinant; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D000263:Adenylate Kinase