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Biological Chemistry

Editor-in-Chief: Brüne, Bernhard

Editorial Board: Buchner, Johannes / Lei, Ming / Ludwig, Stephan / Sies, Helmut / Thomas, Douglas D. / Turk, Boris / Wittinghofer, Alfred


IMPACT FACTOR 2017: 3.022

CiteScore 2017: 2.81

SCImago Journal Rank (SJR) 2017: 1.562
Source Normalized Impact per Paper (SNIP) 2017: 0.705

Online
ISSN
1437-4315
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Volume 382, Issue 2

Issues

Elucidation of the Role of Functional Amino Acid Residues of the Small Sialidase from Clostridium perfringens by Site-Directed Mutagenesis

Reinhard G. Kleineidam / Susanne Kruse / Peter Roggentin / Roland Schauer
Published Online: 2005-06-01 | DOI: https://doi.org/10.1515/BC.2001.038

Abstract

Bacterial sialidases represent important colonization or virulence factors. The development of a rational basis for the design of antimicrobials targeted to sialidases requires the knowledge of the exact roles of their conserved amino acids. A recombinant enzyme of the small (43 kDa) sialidase of Clostridium perfringens was used as a model in our study. Several conserved amino acids, identified by alignment of known sialidase sequences, were altered by sitedirected mutagenesis. All recombinant enzymes were affinitypurified and the enzymatic characteristics were determined. Among the mutated enzymes with modifications in the environment of the 4-hydroxyl group of bound sialic acids, D54N and D54E exhibited minor changes in substrate binding. However, a reduced activity and changes in their pH curves indicate the importance of a charged group at this area. R56K, which is supposed to bind directly to sialic acids as in the homologous Salmonella typhimurium sialidase, showed a 2500-fold reduced activity. The amino acids Asp-62 and Asp-100 are probably involved in catalysis, indicated by reduced activities and altered temperature and pH curves of mutant enzymes. Exchanging Glu-230 with threonine or aspartic acid led to dramatic decreases in activity. This residue and Y 347 are supposed to be crucial for providing a suitable environment for catalysis. However, unaltered pH curves of mutant sialidases exclude their direct involvement in protonation or deprotonation events. These results indicate that the interactions with the substrates vary in different sialidases and that they might be more complex than suggested by mere static Xray structures.

About the article

Published Online: 2005-06-01

Published in Print: 2001-02-12


Citation Information: Biological Chemistry, Volume 382, Issue 2, Pages 313–319, ISSN (Print) 1431-6730, DOI: https://doi.org/10.1515/BC.2001.038.

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