Side Chain Conformation Restriction in the Catalysis of Glycosidic Bond Formation by Leloir Glycosyltransferases, Glycoside Phosphorylases, and Transglycosidases

Abstract

Carbohydrate side chain conformation is an important factor in the control of reactivity at the anomeric center, ie, in the making and breaking of glycosidic bonds, whether chemically or, for hydrolysis, by glycoside hydrolases. In nature glycosidic bond formation is catalyzed out by glycosyltransferases (GTs), glycoside phosphoryases, and transglycosidases. By analysis of 118 crystal structures of sugar nucleotide dependent (Leloir) GTs, 136 crystal structures of glycoside phosphorylases, and 54 crystal structures of transglycosidases bound to hexopyranosides or their analogs at the donor site (-1 site), we determined that most enzymes that catalyze glycoside synthesis, be they GTs, glycoside phosphorylases or transglycosidases, restrict their substrate side chains to the most reactive gauche,gauche (gg) conformation to achieve maximum stabilization of the oxocarbenium ion-like transition state for glycosyl transfer. The galactose series deviates from this trend, with α-galactosyltransferases preferentially restricting their substrates to the second-most reactive gauche,trans (gt) conformation, and β-galactosyltransferases favoring the least reactive trans,gauche (tg) conformation. This insight will help progress the design and development of improved, conformationally-restricted GT inhibitors that take advantage of these inherent side chain preferences.

Keywords: conformational analysis; glycoside hydrolase; glycoside phosphorylase; glycosyltransferase; inhibitor; oxocarbenium ion; transglycosidase.

Figure 1.

The staggered conformations of pyranoside side chains and their approximate population in free solution in the gluco- and galactopyranosides and in the N-acetylneuraminic acids

Figure 2.

Spatial relationships of side chain and C-4 hydroxyl groups with the oxocarbenium π* orbital

Figure 3.

Approximate transition states for inverting and retaining GTs illustrated for D-glucose with the gg side chain conformation.

Figure 4.

Partial structures of (a) UDP-α-galactopyranose (PDB ID 5C1G) and (b) UDP-α-glucopyranose (PDB ID 5C8R) in the donor site of GTB with the tucked under conformation of the pyrophosphate and the side chain restricted to the gt conformation by H-bonding.

Figure 5.

Solution side chain conformations of CMP-N-acetylneuraminic acid (4) and CMP-Kdo (5)

Figure 6.

Partial structures of (a) UDP-5S-GlcNAc donor (PDB ID 4GYY) and (b) a thioglycopeptide product (PDB ID 4GZ3) complexed to human O-GlcNAc transferase

Figure 7.

Partial structures of (a) a cyclodextrin substrate, (b) an inhibitor, and (c) a covalent intermediate in the −1 site of cyclodextrin glycosyltransferases, all with gg side chain conformations.

Figure 8.

Solution conformations of (a) ADP-L-glycero-β-D-manno-heptose (6) and (b) ADP-D-glycero-β-D-manno-heptose (7) and (c) ADP-2-deoxy-2-fluoro-L-glycero-β-D-gluco-heptose bound to E. coli WaaC (PDB ID 2H1H) in the gg conformation.

Figure 9.

Glycosidase inhibitors (a) castanospermine 8 and (b) 1-deoxynojiromcyin 9 and (c) 9 bound to T. maritima β-glucosidase in the gg conformation (PDB ID 2J77)

Scheme 1.

Bound and unbound side chain populations of UDP-2-deoxy-2-fluoro-α-glucose (1) as exemplified by E. coli trehalose-6-phosphate synthase (PDB ID 1UQT)

Scheme 2.

Bound and unbound side chain conformations of UDP-α-N-acetyl glucosamine (2), exemplified in the S. aureus TarP and rabbit N-acetyl glucosaminyltransferase 1 (PDB structures (a) 6H21 and (b) 1FOA)

Scheme 3.

Contrasting binding preferences of α- and β-galactosyltransferases exemplified by (a) cow α-galactosyltransferase and (b) mouse β-galactosyltransferase (PDB structures (a) 2VS5 and (b) 1YRO).

Scheme 4.

Bound and unbound side chain populations of non-hydrolyzable glucose derivatives as exemplified by rabbit muscle glycogen phosphorylase (PDB ID 3L7D)