Active site mutation of the C3-like ADP-ribosyltransferase from Clostridium limosum--analysis of glutamic acid 174.
Clostridium limosum ADP-ribosyltransferase modifies low molecular mass GTP-binding proteins of the Rho subtype family. Here we cloned and sequenced the gene of the transferase and expressed it in Escherichia coli. The gene encodes a protein of 250 amino acids (M(r) = 27,840), with a putative signal peptide of 45 amino acids, that shows about 60-65% identity with C3 transferases from Clostridium botulinum. The mature C. limosum transferase was expressed as a maltose-binding fusion protein in E. coli and purified to apparent homogeneity. To study the functional role of Glu174 of C. limosum transferase, which was recently photoaffinity-labeled with [carbonyl-14C]NAD [Jung, M., et al. (1993) J. Biol. Chem. 268, 23215-23218], two mutants E174D and E174Q were constructed by a polymerase chain reaction-based system. The E174D and E174Q mutants showed a dramatic decrease in kcat, but no major changes in Km,NAD. Furthermore, replacement of Glu174 by aspartic acid and glutamine largely reduced and completely blocked UV-induced incorporation of [carbonyl-14C]NAD into the transferase. The data indicate that Glu174 is an active site residue of C. limosum transferase.
Böhmer
J
J
Institut für Pharmakologie und Toxikologie der Universität des Saarlandes, Homburg, Germany.
Jung
M
M
Sehr
P
P
Fritz
G
G
Popoff
M
M
Just
I
I
Aktories
K
K
D036002:ADP Ribose Transferases / Q000737:chemistry* / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D015139:Blotting, Southern; D001905:Botulinum Toxins; D003001:Cloning, Molecular; D003013:Clostridium / Q000201:enzymology*; D017931:DNA Primers; D004269:DNA, Bacterial / Q000032:analysis / Q000378:metabolism; D004926:Escherichia coli; D018698:Glutamic Acid; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009244:NAD+ Nucleosidase / Q000378:metabolism; D017354:Point Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D004269:DNA, Bacterial; D011994:Recombinant Proteins; D018698:Glutamic Acid; D036002:ADP Ribose Transferases; C063007:exoenzyme C3, Clostridium botulinum; D009244:NAD+ Nucleosidase; D001905:Botulinum Toxins