DPH5, a methyltransferase gene required for diphthamide biosynthesis in Saccharomyces cerevisiae.
A mutant of Saccharomyces cerevisiae defective in the S-adenosylmethionine (AdoMet)-dependent methyltransferase step of diphthamide biosynthesis was selected by intracellular expression of the F2 fragment of diphtheria toxin (DT) and shown to belong to complementation group DPH5. The DPH5 gene was cloned, sequenced, and found to encode a 300-residue protein with sequence similarity to bacterial AdoMet:uroporphyrinogen III methyltransferases, enzymes involved in cobalamin (vitamin B12) biosynthesis. Both DPH5 and AdoMet:uroporphyrinogen III methyltransferases lack sequence motifs commonly found in other methyltransferases and may represent a new family of AdoMet:methyltransferases. The DPH5 protein was produced in Escherichia coli and shown to be active in methylation of elongation factor 2 partially purified from the dph5 mutant. A null mutation of the chromosomal DPH5 gene did not affect cell viability, in agreement with other studies indicating that diphthamide is not required for cell survival. The dph5 null mutant survived expression of three enzymically attenuated DT fragments but was killed by expression of fully active DT fragment A. Consistent with these results, elongation factor 2 from the dph5 null mutant was found to have weak ADP-ribosyl acceptor activity, which was detectable only in the presence of high concentrations of fragment A.
Mattheakis
L C
LC
Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115.
Shen
W H
WH
Collier
R J
RJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D015139:Blotting, Southern; D002384:Catalysis; D003001:Cloning, Molecular; D004271:DNA, Fungal; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D005816:Genetic Complementation Test; D006639:Histidine / Q000031:analogs & derivatives* / Q000096:biosynthesis; D008745:Methylation; D008780:Methyltransferases / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D016415:Sequence Alignment
D004271:DNA, Fungal; D005656:Fungal Proteins; D029701:Saccharomyces cerevisiae Proteins; D006639:Histidine; C027527:diphthamide; D008780:Methyltransferases; C076235:DPH5 protein, S cerevisiae