FellowMgr., PhD. Stanislav Kozmon
Project NameAb initio molecular dynamics study of the glycosyltransferase reaction mechanism as a guide to inhibitors design
Host organisationInstitute of Chemistry
Duration of the project01.07.2015 - 31.12.2018

Glycosyltransferases (GT’s) are indispensable to cellular life in eukaryotes by producing glycan linkages with a unique contribution to the development and function of physiological systems in the context of living organisms. Also connections between GT’s and mammalian disease processes are being made recently. The catalytic mechanism of glycosyltransferases is unclear and high-level QM calculations are used to gain some insight into these enzymatic reactions. The state-of-art hybrid QM/MM calculations and QM/MM molecular dynamics calculations are used to investigate the catalytic mechanism of glycosyltransferases. The N-acetylglucosaminyltransferase V and O-GlcNAc transferase reaction mechanism is studied. Detailed knowledge or the reaction mechanism is necessary for the future inhibitor design. Known connection between GT’s activity and many serious diseases implicates that inhibitors of these enzymes have a great therapeutic potential. A new generation of inhibitors based on enzymatic reaction transition state mimetic promising high inhibition.

Project Summary with Interim Results

One part of the project was focused on the GnT-V glycosyltransferase homology model preparation. The enzyme UDP-N-acetylglucosamine: α-D-mannoside β-1-6 N-acetylglucosaminyltransferase V (GnT-V) catalyzes the transfer of N-acetylglucosamine (GlcNAc) from the UDP-GlcNAc donor to the α-1-6-linked mannose of the trimannosyl core structure of glycoproteins to produce the β1-6-linked branching of N-linked oligosaccharides. β1-6-GlcNAc-branched N-glycans are associated with cancer growth and metastasis. Therefore, the inhibition of GnT-V represents a key target for anti-cancer drug development. However, there is a lack of any information on the three-dimensional structure of the enzyme and the binding characteristics of its substrates. We prepared the first 3-D structure of GnT-V as a result of homology modeling. Various alignment methods, docking the donor and acceptor substrates, and molecular dynamics simulation were used to construct seven homology models of GnT-V and characterize the binding of its substrates. The best homology model is consistent with available experimental data. The three-dimensional model, the structure of the enzyme catalytic site and binding information obtained for the donor and acceptor can be useful in studies of the catalytic mechanism and design of inhibitors of GnT-V.

 Figure: Homology model of the GT-B domain (214-623) of GnT-V with labeled secondary structure elements.

Reaction mechanism of the OGT transferase with modified substrates is another subject of the study within this project. The OGT is UDP-N-acetylglucosamine:polypeptide β-N- acetylglucosaminyltransferase, which adds O-linked GlcNAc residue to serine or threonine residues. We prepared QM/MM models of the OGT ternary complex with UDP-GlcNAc, UDP-5S-GlcNAc as sugar donors and with serine and amino-alanine as carbohydrate acceptors. We prepared models for all possible presented reaction mechanisms. As a first step, we ran a set of the QM/MM metadynamics simulations to observe the preliminary free energy reaction surface.

Based on the transition state structure of the GnT-I inverting glycosyltransferase we suggested several basic scaffolds mimicking the TS structure. From the public databases of the available fragments, we prepared the structural database of the fragments, which will be used to modify basic proposed scaffold. The database contains around 40 000 different fragments.