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R Antoine

Publications and source records attributed to R Antoine.

24 records · Page 2Linked to original sources
PubMed1994

The NAD-glycohydrolase activity of the pertussis toxin S1 subunit. Involvement of the catalytic HIS-35 residue.

Pertussis toxin is a member of ADP-ribosylating bacterial toxins that are capable of catalyzing the cleavage of the N-glycosidic bond of NAD+ and the transfer of its ADP-ribose moiety to G proteins. The catalytic S1 subunit of pertussis toxin uses signal transducing G proteins as acceptor substrates but can also catalyze the transfer of the ADP-ribose moiety to water in the absence of G proteins. Site-directed mutagenesis followed by kinetic analyses of truncated soluble mutant proteins revealed that His-35 of S1 is a catalytic residue because alterations of this residue affect the turnover rate of NAD-glycohydrolysis by approximately two orders of magnitude without significantly affecting substrate binding. Replacement of the imidazole of His-35 by the side chain of glutamine maintained the highest residual activity. The pH dependence of the enzyme activity showed only slight variations over the experimental range with an optimum at pH 7.5 and an approximate pKa of 6.5 to 7. This pH dependence was abolished by the Gln substitution, which still retained significant activity, suggesting that His-35 probably does not act as a true base but rather as a proton acceptor. Direct catalytic roles for several other residues were ruled out. Ser-52 substitutions resulted in slight alterations of both kcat and Km for NAD+ suggesting an involvement in maintaining the local geometry of the active site rather than a direct role in catalysis for this residue. Kinetic studies on mutants with substitutions of Ser-40 indicate a role in NAD+ binding for this residue. In conjunction with previous findings, these studies suggest that the NAD-glycohydrolase activity of S1 utilizes 2 catalytic residues, His-35 and the previously identified Glu-129. The enzyme mechanism could therefore proceed through an activation by polarization of the acceptor substrate water or G protein by His-35, and the stabilization of an oxocarbonium-like transition state intermediate by Glu-129.

Amino Acid Sequence↗
PubMed1993

Evidence for a catalytic role of glutamic acid 129 in the NAD-glycohydrolase activity of the pertussis toxin S1 subunit.

The S1 subunit of pertussis toxin is an ADP-ribosyl-transferase capable of transferring the ADP-ribose moiety of NAD+ to nucleotide-binding signal-transducing proteins of the Gi/G(o) family. In the absence of G proteins, the enzyme also catalyzes the hydrolysis of NAD+. Glu-129 was previously shown to be critical for both enzymatic activities. In this study, site-directed mutagenesis was used to make the conservative substitution of aspartate for Glu-129. The recombinant wild type and mutant proteins were purified to near homogeneity and used for enzymatic analyses. Kinetic experiments showed that the kcat of the mutant protein was about 200 times less than that of the wild type enzyme, whereas the Km for NAD+ of the two proteins were very similar, suggesting that Glu-129 is a catalytic residue for the NAD-glycohydrolase reaction of S1. This hypothesis was confirmed by a less than 2-fold change in Kd as measured by fluorescence quenching studies, indicating that the binding of NAD+ is not affected in the mutant protein in any important way. In another experiment, the replacement of Glu-129 by cysteine resulted in a disulfide bridge between Cys-129 and Cys-41 in rS1d-E129C, suggesting that the folding of the polypeptide chain is such that the catalytic Glu-129 residue is close to the amino-terminal NAD-binding site of S1. These findings imply that Glu-129 plays a key role in catalysis of the NAD-glycohydrolase reaction, possibly by electrostatically stabilizing a cationic transition state intermediate, or by serving as a general base to deprotonate the ADP-ribosyl acceptor substrates.

Base Sequence↗
PubMed1992

Isolation and molecular characterization of a novel broad-host-range plasmid from Bordetella bronchiseptica with sequence similarities to plasmids from gram-positive organisms.

A 2.6 kb plasmid, named pBBR1, was isolated from Bordetella bronchiseptica S87. After insertion of an antibiotic resistance marker, this plasmid could be transferred into Escherichia coli, Bordetella pertussis, B. bronchiseptica, Vibrio cholerae, Rhizobium meliloti, and Pseudomonas putida by transformation or conjugation. Conjugation was possible only when the IncP group transfer functions were provided in trans. As shown by incompatibility testing, pBBR1 does not belong to the broad-host-range IncP, IncQ or IncW groups. DNA sequence analysis revealed two open reading frames: one was called Rep, involved in replication of the plasmid, and the other, called Mob, was involved in mobilization. Both the amino-terminal region of Mob and its promoter region show sequence similarities to Mob/Pre proteins from plasmids of Gram-positive bacteria. In spite of these sequence similarities, pBBR1 does not replicate via the rolling-circle mechanism commonly used by small Gram-positive plasmids. We therefore speculate that pBBR1 may combine a mobilization mechanism of Gram-positive organisms with a replication mechanism of Gram-negative organisms. Determination of the plasmid copy number in E. coli and B. pertussis indicated that pBBR1 has a rather high copy number, which, in conjunction with its small size and broad host range, renders it particularly interesting for studies of broad-host-range replicons and for the development of new cloning vectors for a wide range of Gram-negative bacteria.

Amino Acid Sequence↗
PubMed1992

Tarsal tunnel syndrome secondary to neurilemmoma.

Neurilemmoma is an uncommon cause of tarsal tunnel syndrome, and no other cases of this disorder secondary to a lesion at the bifurcation of the medial and lateral plantar nerves have been described. In this case, a 2 x 0.5 x 1-cm mass was palpable on physical examination, and, after failed conservative treatment, surgical excision brought prompt relief of symptoms.

Adult↗
PubMed1990

Roles of the disulfide bond and the carboxy-terminal region of the S1 subunit in the assembly and biosynthesis of pertussis toxin.

A Bordetella pertussis expression system was developed to analyze the structure-function relationship, in vivo assembly, and biosynthesis of pertussis toxin. The toxin structural gene was first deleted from the B. pertussis chromosome; into the resulting B. pertussis strain the toxin gene was introduced on a low-copy-number, broad-host-range plasmid. The amount of pertussis toxin produced and secreted with this expression system was in the same order of magnitude as that produced by B. pertussis Tohama I, indicating that although the plasmid may be present in more than one copy per cell, overproduction of the toxin was not achieved in B. pertussis. Expression of mutant pertussis toxin genes in which the codon for Cys-41 was deleted or altered or in which the carboxy-terminal region was deleted showed that both the single intrachain disulfide bond and the carboxy-terminal region of S1 are essential for the stable expression, assembly, and secretion of S1. On the other hand, the B oligomer was efficiently secreted in the culture medium in the absence of the S1 subunit. The secreted B oligomer contained S2, S3, and S4 subunits as evidenced by enzyme-linked immunosorbent assay and was fully functional with respect to haptoglobin binding. Furthermore, the deletion of the hydrophobic carboxy-terminal region has a drastic effect on S1 subunit solubility; however, inclusion of the hydrophobic region was not sufficient for assembly and secretion, indicating that other interactions involving amino acids beyond residue 207 of the S1 subunit are also required.

Amino Acid Sequence↗