Redesign of the substrate-binding site of hen egg white lysozyme based on the molecular evolution of C-type lysozymes.
On the basis of the molecular evolution of hen egg white, human, and turkey lysozymes, three replacements (Trp62 with Tyr, Asn37 with Gly, and Asp101 with Gly) were introduced into the active-site cleft of hen egg white lysozyme by site-directed mutagenesis. The replacement of Trp62 with Tyr led to enhanced bacteriolytic activity at pH 6.2 and a lower binding constant for chitotriose. The fluorescence spectral properties of this mutant hen egg white lysozyme were found to be similar to those of human lysozyme, which contains Tyr at position 62. The replacement of Asn37 with Gly had little effect on the enzymatic activity and binding constant for chitotriose. However, the combination of Asn37----Gly (N37G) replacement with Asp101----Gly (D101G) and Trp62----Tyr (W62Y) conversions enhanced bacteriolytic activity much more than each single mutation and restored hydrolytic activity toward glycol chitin. Consequently, the mutant lysozyme containing triple replacements (N37G, W62Y, and D101G) showed about 3-fold higher bacteriolytic activity than the wild-type hen lysozyme at pH 6.2, which is close to the optimum pH of the wild-type enzyme.
Kumagai
I
I
Department of Industrial Chemistry, Faculty of Engineering, University of Tokyo, Japan.
Sunada
F
F
Takeda
S
S
Miura
K
K
D000117:Acetylglucosamine / Q000378:metabolism; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000235:genetics; D000818:Animals; D001419:Bacteria / Q000187:drug effects; D005075:Biological Evolution; D002645:Chickens; D002686:Chitin / Q000031:analogs & derivatives / Q000378:metabolism; D004529:Egg White; D005260:Female; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009113:Muramidase / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D012689:Sequence Homology, Nucleic Acid; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
D000596:Amino Acids; D002686:Chitin; C031610:glycolchitin; D009113:Muramidase; D000117:Acetylglucosamine