Crystal structures of three complexes between chito-oligosaccharides and lysozyme from the rainbow trout. How distorted is the NAG sugar in site D?

Like all c-type lysozymes, those from rainbow trout act as 1,4-β-acetyl-muramidases to destroy bacteria by cleaving the polysaccharide chains of alternating N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) units in the cell walls. Lysozymes also hydrolyse chitin, the analogous N-acetylglucosamine polymer. The rainbow trout enzymes have been shown to be particularly effective in bacterial defence. We have determined the crystal structures of three complexes between rainbow trout lysozyme (RBTL) and the chito-oligosaccharides (NAG)₂, (NAG)₃ and (NAG)₄ to resolutions of 1.8, 2.0 and 1.6 Å, respectively. Crystals of these complexes were obtained by co-crystallization, and intensity data were collected on a FAST area detector system. Refinement and model building gave final R values of 16.6, 15.9 and 16.5% for the di-, tri- and tetrasaccharide complexes, respectively. The results show that the chito-oligosaccharides bind to sites A, B and C as previously observed for complexes between the hen egg-white lysozyme (HEWL) and a variety of saccharides. The NAG ring in site D is not bound so deeply and is only slightly distorted towards a half-chair conformation as observed for the equivalent NAM residue in HEWL. From our results, there is reason to question the position and the degree of strain of the D saccharide and the mode of binding and importance of two saccharides in sites E and F for correct orientation of sugar D and effective hydrolysis of a productive substrate–lysozyme complex. Simple model building study from our structures implies a `left-sided' binding mode of (NAG)₆ in the lower part of the active site of RBTL.