Characterization of Vis Toxin, a Novel ADP-Ribosyltransferase from Vibrio splendidus.
Vis toxin was identified by a bioinformatics strategy as a putative virulence factor produced by Vibrio splendidus with mono-ADP-ribosyltransferase activity. Vis was purified to homogeneity as a 28 kDa single-domain enzyme and was shown to possess NAD(+)-glycohydrolase [KM(NAD(+)) = 276 ± 12 μM] activity and with an R-S-E-X-E motif; it targets arginine-related compounds [KM(agmatine) = 272 ± 18 mM]. Mass spectrometry analysis revealed that Vis labels l-arginine with ADP-ribose from the NAD(+) substrate at the amino nitrogen of the guanidinium side chain. Vis is toxic to yeast when expressed in the cytoplasm under control of the CUP1 promotor, and catalytic variants lost the ability to kill the yeast host, indicating that the toxin exerts its lethality through its enzyme activity. Several small molecule inhibitors were identified from a virtual screen, and the most potent compounds were found to inhibit the transferase activity of the enzyme with Ki values ranging from 25 to 134 μM. Inhibitor compound M6 bears the necessary attributes of a solid candidate as a lead compound for therapeutic development. Vis toxin was crystallized, and the structures of the apoenzyme (1.4 Å) and the enzyme bound with NAD(+) (1.8 Å) and with the M6 inhibitor (1.5 Å) were determined. The structures revealed that Vis represents a new subgroup within the mono-ADP-ribosyltransferase toxin family.
Department of Molecular and Cellular Biology, University of Guelph , Guelph, Ontario, Canada N1G 2W1.
Ravikiran
R
Ravulapalli
Department of Molecular and Cellular Biology, University of Guelph , Guelph, Ontario, Canada N1G 2W1.
Miguel R
MR
Lugo
Department of Biology, York University , Toronto, ON, Canada M3J 1P3.|Department of Biochemistry, University of Toronto , Toronto, ON, Canada M5S 1A8.|Campbell Family Institute for Cancer Research, Princess Margaret Hospital , Toronto, ON, Canada M5G 1L7.
Roland
R
Pfoh
Department of Molecular and Cellular Biology, University of Guelph , Guelph, Ontario, Canada N1G 2W1.
Danielle
D
Visschedyk
Department of Molecular and Cellular Biology, University of Guelph , Guelph, Ontario, Canada N1G 2W1.
Amanda
A
Poole
Computational Biology Center, Memorial Sloan-Kettering Cancer Center , New York, New York 10065, United States.|Department of Systems Biology, Harvard Medical School , Boston, Massachusetts 02115, United States.
Robert J
RJ
Fieldhouse
Department of Biochemistry, University of Toronto , Toronto, ON, Canada M5S 1A8.|Campbell Family Institute for Cancer Research, Princess Margaret Hospital , Toronto, ON, Canada M5G 1L7.|Departments of Medical Biophysics and Molecular Genetics, University of Toronto , Toronto, ON, Canada M5S 1A8.
Emil F
EF
Pai
Department of Molecular and Cellular Biology, University of Guelph , Guelph, Ontario, Canada N1G 2W1.
A Rod
AR
Merrill
D036002:ADP Ribose Transferases / Q000737:chemistry* / Q000378:metabolism; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000595:Amino Acid Sequence; D001427:Bacterial Toxins / Q000737:chemistry* / Q000378:metabolism; D018360:Crystallography, X-Ray; D019791:Guanidine / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009244:NAD+ Nucleosidase / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D016415:Sequence Alignment; D014733:Vibrio / Q000737:chemistry / Q000201:enzymology* / Q000378:metabolism
D001427:Bacterial Toxins; D000246:Adenosine Diphosphate Ribose; D036002:ADP Ribose Transferases; D009244:NAD+ Nucleosidase; D019791:Guanidine