Abstract
Escherichia coli heat-labile enterotoxin (LT) and the related cholera toxin exert their effects on eukaryotic cells through the ADP-ribosylation of guanine nucleotide-binding proteins of the adenylate cyclase complex. The availability of the crystal structure for LT has permitted the tentative identification of residues that lie within or are vicinal to a presumptive NAD(+)-binding site and thus may play a role in substrate binding or catalysis. Using a plasmid clone encoding the A subunit of LT, we have introduced substitutions at such potential active-site residues and analyzed the enzymatic properties of the resultant mutant analogs. Enzymatic analyses, employing both transducin and agmatine as acceptor substrates, revealed that substitutions at serine 61, glutamic acid 110, and glutamic acid 112 resulted in reduction of enzyme activity to < 10% of wild-type levels. Kinetic analyses indicated that alteration of these sites affected the catalytic rate of the enzyme and had little or no effect on the binding of either NAD+ or agmatine. Of the mutant analogs analyzed, only glutamic acid 112 appeared to represent an essential catalytic residue as judged by the relative effects on kcat and kcat/Km. The results provide formal evidence that glutamic acid 112 of the A subunit of LT represents a functional homolog or analog of catalytic glutamic acid residues that have been identified in several other bacterial ADP-ribosylating toxins and that it may play an essential role in rendering NAD+ susceptible to nucleophilic attack by an incoming acceptor substrate.
Highlights
From the Laboratory of Intracellular Parasites, Rocky Mountain Laboratories, NIAID, National Institutes of Health, Hamilton, Montana 59840
The results provide formal evidence that glutamic acid 112 of the A subunit of labile enterotoxin (LT) represents a functional homolog or analog of catalytic glutamic acid residues that have been identified in several other bacterial ADP-ribosylating toxins and that it may play an essential role in rendering NAD؉ susceptible to nucleophilic attack by an incoming acceptor substrate
The other His residue that may be of importance is His-44, which is located in a -sheet that is in close proximity to the NADϩ-binding site and has been proposed to be functionally equivalent to a His residue (His-35) that appears to play a role in NADϩ binding in pertussis toxin (PT) [2, 24]
Summary
Materials—DNA-modifying enzymes were purchased from New England Biolabs Inc. and Life Technologies, Inc. and were used according to the recommendations of the supplier. [adenylate-32P]NADϩ (20 –30 Ci/mmol) and [carbonyl-14C]NADϩ (41–54 mCi/mmol) were obtained from DuPont NEN and Amersham Corp., respectively. Recombinant bovine ADP-ribosylation factor I (rARF-I) was purified from E. coli BL21(DE3) harboring plasmid pOW12 [25] as described [13]. Bacterial Strains and Expression Plasmids—E. coli DH5␣ was obtained from Life Technologies, Inc., and phagemid pTZ18R was obtained from Pharmacia Biotech Inc. The construction of recombinant expression plasmid pTZrLTA has been described in detail [13]. Plasmid DNA was extracted from 5 to 10 electrotransformants and subjected to DNA sequence analysis to confirm the presence of the desired mutations and the fidelity of the amplifications. The ability to ADP-ribosylate the ␣-subunit of transducin in bovine ROS membranes was assayed using the recombinant subunits at a final concentration of 25 g/ml as described [13]. Immunoblotting, using a combination of monoclonal and rabbit polyclonal anti-A subunit antibodies and horseradish peroxidase-linked anti-mouse IgG and antirabbit IgG, was done as described previously [12]
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