Figure 1.
Synthesis of glycopeptides using oligosaccharide oxazolines as sugar donors.
(A) Mimic of oxazoline ion intermediate, Man3GlcNAc-oxazoline. (B) Endo-A catalyzed glycopeptide synthesis by using Man3GlcNAc-oxazoline. (C) An Endo-A inhibitor, Man3GlcNAc-thiazoline.
Figure 2.
(A) Diagrammatic representation of Endo-A. Amino acids 1–350 make up Domain 1 (blue), segments 351–362 and 387–524 make up Domain 2 (orange), while segments 363–386 and 525–611 make up Domain 3 (red). (B) Stereo image of Endo-A. Man3GlcNAc-thiazoline moiety is shown as sticks. (C) A surface electrostatic potential representation of Endo-A showing the Man3GlcNAc-thiazoline moiety sitting inside the active site cleft.
Figure 3.
Stereo images of Endo-A in complex with GlcNAc-Asn (panel A) and Man3GlcNAc-thiazoline (panel B).
The ligand omitted |Fo|-|Fc| αcalc electron density map, calculated using a 3.5 Å resolution data set, shown in blue, was contoured at 2.5σ.
Figure 4.
(A) N171, E173 and Y299 are critical for catalysis. Amino acids surrounding the carbohydrate moiety are shown as sticks. The distances are shown in dashed lines. The GlcNAc-Asn moiety is shown as sticks. (B) W216 and W244 are “gate-keeping” the active site by sterically regulating access to the active site by the acceptor. The side chain of W244 moves during transglycosylation; the distance between the two Trp is much wider and allows passage of an acceptor into the active site. Trp and GlcNAc-Asn are shown as sticks. (C) The gate is “closed” in the structure of free protein (not shown) and Man3GlcNAc-thiazoline bound Endo-A (shown as sticks).
Figure 5.
Transglycosylation activity of Endo-A mutants.
Time course of transglycosylation reaction of wildtype Endo-A (squares) and the three mutants, N171A (circles), Y205F (triangles), and Y299F (diamonds). The reactions were carried out using a chitinase-coupled assay, in which Man9GlcNAc2Asn-Fmoc was used as the donor substrate and 4MU-GlcNAc as the acceptor, 0.25 µg of each enzyme was used in a 50 µl system.
Table 1.
Hydrolysis and transglycosylation activities of Endo-A mutants.
Figure 6.
Mechanism of Endo-A mediated catalysis.
(A) Substrate surrounded by critical amino acids - N171, E173 and Y205. The nucleophilic oxygen (O) is marked with *. (B) Intramolecular nucleophilic attack. (C) Formation of an oxazoline ion intermediate. (D) Second nucleophilic attack on the intermediate. (E) Synthesis of a new glycosidic bond. R' could be a GlcNAc molecule of an acceptor during transglycosylation.
Table 2.
Data collection and refinement statistics.