Crystal structures of g-type lysozyme from Atlantic cod shed new light on substrate binding and the catalytic mechanism

Abstract

Crystal structures of Atlantic cod lysozyme have been solved with and without ligand bound in the active site to 1.7 and 1.9 A resolution, respectively. The structures reveal the presence of NAG in the substrate binding sites at both sides of the catalytic Glu73, hence allowing the first crystallographic description of the goose-type (g-type) lysozyme E-G binding sites. In addition, two aspartic acid residues suggested to participate in catalysis (Asp101 and Asp90) were mutated to alanine. Muramidase activity data for two single mutants and one double mutant demonstrates that both residues are involved in catalysis, but Asp101 is the more critical of the two. The structures and activity data suggest that a water molecule is the nucleophile completing the catalytic reaction, and the roles of the aspartic acids are to ensure proper positioning of the catalytic water.

Crystal Structures of the Catalytic Domain of a Novel Glycohydrolase Family 23 Chitinase from Ralstonia sp. A-471 Reveals a Unique Arrangement of the Catalytic Residues for Inverting Chitin Hydrolysis

“…G-type lysozymes have a highly conserved glutamic acid residue, Glu 73 , as a general acid (25)(26)(27). In addition, Helland et al (28) , respectively (17), which are the best candidates for catalytic residues. However, this is only a suggestion from sequence comparison, and the catalytic residues of the unusual chitinase, Ra-ChiC, remains to be elucidated by the structural and mutation analyses.…”

Structural Insights on the Bacteriolytic and Self-protection Mechanism of Muramidase Effector Tse3 in Pseudomonas aeruginosa

“…There are mainly three types of lysozymes: chicken-type, goose-type, and lysozyme from T4 bacteriophage (36,38,39). The goose-type lysozymes adopt a ␣ϩ␤ fold consisting of seven ␣-helices and three ␤-strands (36).…”

“…The goose-type lysozymes adopt a ␣ϩ␤ fold consisting of seven ␣-helices and three ␤-strands (36). In Tse3, the central helix ␣14 contains the highly conserved glutamic acid Glu-250 (Glu-73 in goose-type lysozyme, Glu-478 in Slt70, and Glu-64 in MltE), which is proposed to act as a general-acid catalyst (36).…”

“…The goose-type lysozymes adopt a ␣ϩ␤ fold consisting of seven ␣-helices and three ␤-strands (36). In Tse3, the central helix ␣14 contains the highly conserved glutamic acid Glu-250 (Glu-73 in goose-type lysozyme, Glu-478 in Slt70, and Glu-64 in MltE), which is proposed to act as a general-acid catalyst (36). Another ␤-hairpin also exhibits high conservation, the conserved glutamine Gln-280 of Tse3 (Gln-99 in goose-type lysozyme, Gln-496 in Slt70, and Gln-82 in MltE) is located on this ␤-hairpin and coordinates with Ca 2ϩ cations (Fig.…”

“…Another ␤-hairpin also exhibits high conservation, the conserved glutamine Gln-280 of Tse3 (Gln-99 in goose-type lysozyme, Gln-496 in Slt70, and Gln-82 in MltE) is located on this ␤-hairpin and coordinates with Ca 2ϩ cations (Fig. 2B), although no metal ion binding was reported in these homologues (35)(36)(37).…”