Molecular Basis of Glycolipid Biosynthesis at the Membrane Surface

David Albesa-Jové^1,2,3 and Marcelo E. Guerin^1,2,3

1.  Biophysics Unit, Consejo Superior de Investigaciones CientíficasUniversidad del País Vasco (CSIC, UPV), Leioa, Bizkaia 48940, Spain.

2.  Departamento de Bioquímica, Universidad del País Vasco, Spain.

3.  IKERBASQUE, Basque Foundation for Science, Bilbao, Spain

Phosphatidylinositol mannosides (PIMs), lipomannan and lipoarabinomannan (1,2,3) glycolipids are key structural elements and virulence factors of Mycobacterium tuberculosis. In the last few years we have been studding the molecular mechanisms of enzymes involved in their synthesis (4,5). These enzymes are essential integral or peripheral membrane proteins that have evolved to catalyse transfer reactions between hydrophobic and hydrophilic substrates residing within the chemically distinct environments defined by the phospholipid-based membranes and the aqueous lumens of the cytoplasm or periplasm. One mechanistic solution for these enzymes to combine hydrophobic and hydrophilic substrates is to catalyse their reactions at the membrane-water interface. In this way, both hydrophobic and hydrophilic substrates remain in their original environment during catalysis. Several enzymes participating in this pathway are glycosyltransferases (GTs). Glycosyl transfer can proceed with either ‘inversion’ or ‘retention’ of the anomeric configuration with respect to the reaction substrates and products. The elucidation of the catalytic mechanism of retaining GTs has been the matter of strong debate and remains a major challenge. We will present the first native ternary complex of a GT (6), in the presence of the sugar donor UDP-Glc, the acceptor substrate and divalent cation cofactor, in a productive mode for catalysis. In combination with structural, chemical, enzymatic, molecular dynamics and quantum-mechanics/molecular-mechanics (QM/MM) calculations, we unravel its catalytic mechanism, providing a strong experimental support for a front-side, substrate assisted S_Ni-type reaction.