X-ray crystallography of the binding of the bacterial cell wall trisaccharide NAM-NAG-NAM to lysozyme

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Abstract
Hen egg white lysozyme was the first enzyme whose structure was determined by X-ray crystallography1. The proposed mechanism2–4 based on this structure involves the distortion of the saccharide residue (2-acetamido-2-deoxy-D-muramic acid, NAM) in the natural substrate5 (an alternating β(1→4) linked oligomer of 2-acetamido-2-deoxy-D-glucose (NAG) and NAM residues) bound to site D in the binding cleft. The importance of substrate distortion has prompted numerous enzymatic6, chemical17, theoretical8–12, and physical13 studies, but there is little direct crystallographic evidence on the conformation of a NAM residue bound at site D. We now present the X-ray structure of the non-hydrolysed13 trisaccharide NAM-NAG-NAM bound in subsites B, C, D. Our interpretation of the 2.5-Å resolution difference map does not involve distortion of this residue in site D. Comparison with the structure of the δ-lactone derived from tetra N-acetylchitotetraose ((NAG)3NAL) bound to lysozyme14 suggests we may be looking at a Michaelis complex.