1NCBR, Masaryk University, Kotlarska 2, 611 37 Brno, Czech republic
2CERMAV – CNRS, 601 rue de la Chimie, BP 53, 38041 Grenoble, France
The glycosyltransferases catalyze the transfer of glycosyl moieties from a donor sugar to an acceptor. In most cases, the donor is a nucleoside phosphosugar and the acceptor a hydroxyl group of another sugar, a lipid, or another component of glycoconjugates. The catalytic mechanism of the glycosyltransferases, however, still remains a mystery. The glycosyltransferases are classified as either retaining or inverting, depending on the stereochemical outcome of the reaction catalyzed.
The galactosyltransferase LgtC   from Neisseria meningitidis is a retaning glycosyltransferase catalyzing a key step in the biosynthesis of lipooligosaccharide structure by transferring a-d-galactose from UDP-galactose to a terminal lactose. The comprehension of the catalytic mechanism is an essential precondition to be able to design an effective inhibitor of this enzyme, and to find an effective drug against the bacterial pathogen in this way. We report here the first molecular dynamics simulations of LgtC in water environment and in the presence of its substrates. The analyses of the trajectories provide a first insight on the catalytic mechanism of this glycosyltransferase.
The starting coordinates were taken from the X-ray structure of LgtC galactosyltransferase with donor and acceptor sugar analogs, which is deposited in the Protein Databank under the code 1GA8. The original pdb file had to be modified, as it contained coordinates for 278 amino acids, the donor analog UDP-2-deoxy-2-fluorogalactose and the acceptor analog 4-deoxylactose. The four missing residues 218-221 were added in Sybyl 6.7 using protein loop search. The donor analog was replaced with UDP-galactose (native donor), and the acceptor analog was replaced with lactose (native acceptor).
Simulations were performed using the AMBER-6.0 program package with several new parameters especially developped for nucleotide sugars . All simulations were run with the SANDER. The MD trajectories were analyzed with the CARNAL and PTRAJ.
Four MD simulations were run using the X-ray derived geometry as the starting point. The mobility of two loops essential for the correct functioning of the enzyme were studied together with the stability of coordination of the manganese ion and its role in the binding of substrates.
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2. Ly, H.D., et al., Mechanistic Studies of a Retaining a-Galactosyltransferase from Neisseria meningitidis. Biochemistry 2002, 2002. 41(16): p. 5075-5085.
3. Petrová, P., J. Koča, and A. Imberty, Potential Energy Hypersurfaces of Nucleotide Sugars: Ab Initio Calculations, Force-Field Parametrization, and Exploration of the Flexibility. J. Am. Chem. Soc., 1999. 121(23): p. 5535-5547.