Quantum-chemical insight into the reaction mechanism of polypeptide UDP-GalNAc transferase 2, a retaining glycosyltransferase


Tomáš Trnka1, Stanislav Kozmon1, Igor Tvaroška1,2, Jaroslav Koča1

 

National Centre for Biomolecular Research, Faculty of Science, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic

Institute of Chemistry, Centre for Glycomics, Slovak Academy of Sciences, Dúbravska cesta 9, 845 38 Bratislava, Slovakia

ttrnka@mail.muni.cz


Protein glycosylation is thought to be main means of cell recognition. Misregulation of the cascade of glycosyltransferases is related to many diseases with the most prominent example being cancer.[1] There is thus significant scientific interest in the reaction mechanisms of glycosyltransferases because knowledge of transition state structures would enable targeted design of selective inhibitors usable as potential drugs. However, reaction mechanism of the configuration-retaining group of glycosyltransferases hasn't been explained yet.[2]

For his reason we have chosen a retaining glycosyltransferase – polypeptide UDP-GalNAc transferase (ppGalNAcT) – as the subject of our quantum-chemical study. This enzyme catalyses the transfer of N-acetylgalactosamine moiety onto protein serine or threonine hydroxyls, forming the first bond of the so-called O-linked glycosylation pathway. Increased activity of this enzyme has been found to enable metastasis of breast and colorectal cancer.[3] These enzymes form a large family with twenty isoforms described in humans to date. Even though all ppGalNAc transferases exhibit strong mutual structural similarity, there are significant differences in their preference for protein substrates and glycosylation site location.[2]

Thanks to the availability of high-resolution X-ray structures of three members of the ppGalNAcT family (human transferases 2 and 10, murine transferase 1) we have been able to successfully mount a quantum chemistry study of the human ppGalNAcT2, leveraging information on substrate positioning in active site from the ppGalNAcT10. We are using a hybrid quantum mechanics/molecular mechanics approach using density functional theory on the BP86/TZP level for the important part of the active site. Structures in reactant and product energy minima have been successfully obtained, enabling a potential energy surface scan to find the locations of transition state candidates. Results clearly show that proton transfer between the acceptor hydroxyl moiety and the donor phosphate plays a crucial role in enabling the reaction to take place. The 2D energy map suggests that the reaction proceeds via a two-step mechanism with formation of a carbocation intermediate and its subsequent nucleophilic trapping by the acceptor oxygen. However, exact location of the transition states is yet necessary to prove this conclusively.

This work was realized in CEITEC - Central European Institute of Technology with research infrastructure supported by the project CZ.1.05/1.1.00/02.0068 financed from European Regional Development Fund.

The research has been supported by the EU Seventh Framework Programme under the "Capacities" specific programme (Contract No. 286154).

Tomáš Trnka is a Brno Ph.D. Talent Scholarship Holder – Funded by the Brno City Municipality.

The access to the MetaCentrum computing facilities provided under the program "Projects of Large Infrastructure for Research, Development, and Innovations" LM2010005 funded by the Ministry of Education, Youth, and Sports of the Czech Republic is acknowledged.

 

1.       Brockhausen, I.; Biochim. Biophys. Acta, 1999, 1473, 69–95

2.       ten Hagen, K. G.; Fritz, T. A.; Tabak, L. A.; Glycobiology, 2003, 13 (1), 1R–16R

3.       Gill, D. J.; Clausen, H.; Bard, F.; Trends Cell Biol., 2011, 21 (3), 149–158