Knowledge-based homology modelling and experimental determination
of amino acid side chain accessiblity by the laser photo
CIDNP(chemically induced dynamic nuclear polarization) approach in
solution: lessons from the small sialidase of Clostridium
perfringens
- Institut für Physiologische Chemie, Tierärztliche
Fakultät, Ludwig-Maximilians-Universität, Veterinärstr.
13, D-80539 München, Germany
- Bijvoet Center for
Biomolecular Research, University of Utrecht, P. O. Box 80075,
NL-3508 TB Utrecht, The Netherland
- Zentrale Spektroskopie,
Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, D-69120
Heidelberg, Germany
- Department of Medicinal Chemistry, School of Pharmacy, Tehran
University of Medical Sciences, P. O. Box 14155/6451, Tehran, Iran
- Biochemisch Laboratorium, University of Groningen, Nijenborgh 4,
NL-9747 AG Groningen, The Netherland
- Biochemisches Institut, Christian-Albrechts Universität,
Olshausenstr. 40, D-24098 Kiel, Germany
Abstract
Sialidases (N-acylneuraminosyl-glycohydrolases, EC 3.2.1.18)
hydrolytically cleave alpha-glycosidically bound sialic acids,
derivatives of the amino sugar neuraminic acid. Sialic acids are mostly
found as terminal constituents of oligosaccharides, glycoproteins and
glycolipids in higher animals. The sialidases, however, are widely
distributed not only throughout the metazoan animals of the
deuterostomate lineage, but also among protozoa, viruses, fungi and
bacteria, most of which are unable to produce sialic acid by
themselves. Remarkably, the enzyme is often produced by microorganisms,
which live in close contact with an animal host, whereby the enzyme may
serve as a pathogenicity factor, or as an important tool for
nutrition.
X-ray structure of sialidase of Salmonella typhimurium was used
as the template for homology modeling of Clostridium perfringens
sialidase (CPS). Both FastA and BLAST algorithm indicate high
similarity between these two enzymes. The amino acids located in the
four "Asp boxes" and those of the active site of the enzyme are highly
conserved. Construction of the starting framework, fitting of the CPS
backbone, addition of loop regions and missing side chains, and
preliminary refinement of model were carried out using the Swiss-Model
Automated Protein Modelling service. The generation of hydrogen atoms
and automatic assignment of partial charges of each atom were
accomplished using the INSIGHTII. The structures were then submitted to
an MD simulation using the CVFF force field at a temperature of 300 K
with an equilibration time of 20 ps and production period of 100 ps.
The ten lowest potential energy conformers were selected for further
minimization and surface accessibility calculation of aromatic
residues.
The function of sialidases can be studied with help of mutants
constructed by site-directed mutagenesis. Based on the known three-
dimensional structure of the Salmonella typhimurium sialidase,
amino acids analogous to those that seem to be important for substrate
binding or catalysis, were selected for mutation in CPS. The activity
of some of the mutant sialidases was strongly decreased but the
Km-values were hardly changed.
The side chains of tyrosine, tryptophan and histidine are able to
produce CIDNP (Chemically Induced Dynamic Nuclear Polarization) signals
after laser irradiation in the presence of a suitable radical
pair-generating dye. The CIDNP technique has previously been used for
comparative studies of non-specific and specific interaction between
the lac-repressor headpiece and DNA denatured states of lysozyme as
well as of glycoproteins in glycosylated and deglycosylated form or in
sialylated and desialylated form in solution.
The results from CIDNP experiments with CPS and its mutant forms
indicated significant changes in the pattern of surface accessibility
of aromatic residues of CPS in all of the mutants, which is in complete
agreement with the measured Connolly surfaces of amino acids in the
modeled structures.
* Author to whom correspondence should be addressed.