Conservation of a common motif in enzymes catalyzing ADP-ribose transfer. Identification of domains in mammalian transferases.
Bacterial toxin ADP-ribosyltransferases, e.g. diphtheria toxin (DT) and pertussis toxin, have in common consensus sequences involved in catalytic activity, which are localized to three regions. Region I is notable for a histidine or arginine; region II, approximately 50-75 amino acids downstream, is rich in aromatic/hydrophobic amino acids; and region III, further downstream, has a glutamate and other acidic amino acids. A similar motif was observed in the sequence of the glycosylphosphatidylinositol-linked muscle ADP-ribosyltransferase. Site-directed mutagenesis was performed to verify the role of this motif. Proteins were expressed in rat adenocarcinoma cells, released from the cell with phosphatidylinositol-specific phospholipase C, and quantified with polyclonal antibodies. Transferase His114 in region I aligned with His21 of DT; as with DT, the H114N mutant was active. Aromatic/hydrophobic amino acids (region II) were found approximately 30-50 amino acids downstream of this histidine. Although transferase has a Glu278-Tyr-Ile sequence characteristic of region III in DT, Glu278 was not critical for activity. In an alternative region III containing Glu238-Glu239-Glu240, Glu238 and Glu240 but not Glu239 were critical. Glu240 aligned with critical glutamates in DT, Pseudomonas exotoxin, and C3 transferase. Thus, the mammalian ADP-ribosyltransferases have motifs similar to toxin ADP-ribosyltransferases, suggesting that these sequences are important in ADP-ribose transfer reactions.
Takada
T
T
Pulmonary-Critical Care Medicine Branch, NHLBI, National Institutes of Health, Bethesda, Maryland 20892-1434.
Iida
K
K
Moss
J
J
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002384:Catalysis; D017931:DNA Primers; D018698:Glutamic Acid / Q000378:metabolism; D006639:Histidine / Q000378:metabolism; D008969:Molecular Sequence Data; D011065:Poly(ADP-ribose) Polymerases / Q000737:chemistry* / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D000246:Adenosine Diphosphate Ribose; D018698:Glutamic Acid; D006639:Histidine; D011065:Poly(ADP-ribose) Polymerases