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.
Department of Microbiology, University of California, Los Angeles 90095, USA. berniew@microbio.lifesci.ucla.edu
B J
BJ
Wisnieski
S M
SM
Amini
Y
Y
Tseng
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