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S Chauvaux

Publications and source records attributed to S Chauvaux.

5 recordsLinked to original sources
PubMed1992

Site-directed mutagenesis of essential carboxylic residues in Clostridium thermocellum endoglucanase CelD.

12 carboxyl residues of the Clostridium thermocellum endoglucanase CelD were mutated to alanine. The specific activity of five of the mutated proteins was 1% or less that of wild type. The Ca2+ binding isotherms of these five were similar to those of wild type CelD, consistent with the fact that none of the mutated residues is observed to be directly involved in Ca2+ binding in the three-dimensional structure of the protein (Juy, M., Anit, A. G., Alzuri, P. M., Poljak, R. J., Claeyssens, M., Béguin, P., and Aubert, J.-P., manuscript in preparation) and suggesting that the mutations did not result in gross alterations of the tertiary structure. Analysis of the physico-chemical and enzymatic properties of the five purified mutated proteins and consideration of their position in the three-dimensional structure suggest that carboxyl groups identified may play roles as a general acid catalyst and a source of negative charge in stabilizing a carbonium ion intermediate. Among mutated residues, Glu-555 appears as the most likely candidate for participating in the catalytic mechanism of endoglucanase CelD.

Amino Acid Sequence↗
PubMed1991

Identification of a histidyl residue in the active center of endoglucanase D from Clostridium thermocellum.

Diethylpyrocarbonate modification of endoglucanase D from Clostridium thermocellum, cloned in Escherichia coli, resulted in a rapid but partial (maximally 70-80%) loss of activity. The second-order rate constant of inactivation proved to be exceptionally high (3210 M-1.min-1). A 3-fold reduction of the kcat and a 2-fold increase of the Km for 2'-chloro-4'-nitrophenyl beta-cellobioside were observed. Spectrophotometric analysis indicate the presence of one rapidly (k = 0.45 min-1) and two slower (k = 0.23 min-1) reacting histidyl residues. In the presence of 50 mM methyl beta-cellotrioside, the rate of inactivation was reduced 16-fold, and the kinetics of modification were compatible with the protection of 1 histidyl residue. Since peptide analysis was inconclusive, identification of the critical residue was attempted by site-directed mutagenesis. Each of the 12 histidyl residues present in the endoglucanase D sequence was mutated into either Ala or Ser. Seven of the mutant enzymes had specific activities lower than 50% of the wild-type. Only in the case of the Ser-516 mutant, however, was the residual activity not affected by diethyl pyrocarbonate. These findings suggest an important functional or structural role for His-516 in the wild-type enzyme.

Amino Acid Sequence↗
PubMed1990

Calcium-binding affinity and calcium-enhanced activity of Clostridium thermocellum endoglucanase D.

Clostridium thermocellum endoglucanase D (EC 3.2.1.4: EGD), which is encoded by the celD gene, was found to bind Ca2+ with an association constant of 2.03 x 10(6) M-1. Ca2+ stimulated the activity of EGD towards swollen Avicel by 2-fold. In the presence of Ca2+, the Kd of the enzyme towards p-nitrophenyl-beta-D-cellobioside and carboxymethylcellulose was decreased by 4-fold. Furthermore, Ca2+ increased the half-life of the enzyme at 75 degrees C from 13 to 47 min. Since the 3' sequence of celD encodes a duplicated region sharing similarities with the Ca2+-binding site of several Ca2+-binding proteins, a deleted clone was constructed and used to purify a truncated form of the enzyme which no longer contained the duplicated region. The truncated enzyme was very similar to EGD expressed from the intact gene with respect to activity, Ca2(+)-binding kinetics and Ca2+ effects on substrate binding and thermostability. Thus the latter parameters do not appear to be mediated through the duplicated conserved region.

Amino Acid Sequence↗
PubMed1989

Fluorometric assay for the measurement of viral neuraminidase in influenza vaccines.

Influenza viruses have two surface glycoproteins: haemagglutinin and neuraminidase which are capable of inducing a significant antibody response following vaccination. All neuraminidases from different strains of influenza A and B viruses are able to hydrolyse alpha-ketosidic linkages between N-acetylneuraminic (sialic) acid and other carbohydrates. In this report, the neuraminidase activity was assayed in various influenza vaccines by using a fluorogenic substrate: the sodium salt of 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid. This method was reliable (variation less than 8%) and more sensitive (100 to 1000 times) in less time (incubation time = 15 min) than the Warren assay. Therefore, the method is suitable for the control of the sialidase activity during the processing of influenza vaccines.

Fluorometry↗