Characterization of the deoxyribonuclease and ADP-ribosyltransferase activities of CRM45, a truncated homologue of diphtheria toxin.
CRM45 is a mutant form of diphtheria toxin (DTx) that lacks a 17-kDa carboxyl-terminal segment of the receptor-binding B subunit (DTB). The missing segment is a discrete structural domain of DTB that normally rests against the NAD binding pocket of the enzymically-active A subunit (DTA). Proteolytic cleavage and disulfide bridge reduction in the DTA-DTB linker region of DTx are required for optimal ADP-ribosylation of elongation factor 2 (EF-2). Here, we show that cleaved and uncleaved preparations of X-ray crystal grade CRM45 both exhibit an ADP-ribosyltransferase activity similar to that of cleaved DTx. Crystal-grade preparations of CRM45 also display a potent deoxyribonuclease activity. However, as observed with DTx, cleavage and reduction of CRM45 are not required for expression of this nuclease activity. After SDS-PAGE in a gel that contains DNA embedded in the matrix, renaturable Ca++/Mg(++)-dependent nuclease-active bands co-migrate with intact CRM45 (45 kDa) as well as with the DTA subunit (24 kDa) of CRM45. Because the 45-kDa nuclease-active band is unique to the CRM45 form of DTx, it offers direct proof that this activity is intrinsic to the DTA domain of DTx and its homologues.
Wisnieski
B J
BJ
Department of Microbiology, University of California, Los Angeles 90095, USA. berniew@microbio.lifesci.ucla.edu
Amini
S M
SM
Tseng
Y
Y
D001426:Bacterial Proteins / Q000032:analysis / Q000737:chemistry*; D002118:Calcium; D003851:Deoxyribonucleases / Q000032:analysis*; D004167:Diphtheria Toxin / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D007091:Image Processing, Computer-Assisted; D008274:Magnesium; D015394:Molecular Structure; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors / Q000737:chemistry; D011065:Poly(ADP-ribose) Polymerases / Q000032:analysis*
D001426:Bacterial Proteins; C033445:CRM45 fragment of diphtheria toxin; D004167:Diphtheria Toxin; D004220:Disulfides; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors; D011065:Poly(ADP-ribose) Polymerases; D003851:Deoxyribonucleases; D008274:Magnesium; D002118:Calcium