Crystal structures of K33 mutant hen lysozymes with enhanced activities.
Using random mutagenesis, we previously obtained K33N mutant lysozyme that showed a large lytic halo on the plate coating Micrococcus luteus. In order to examine the effects of mutation of K33N on enzyme activity, we prepared K33N and K33A mutant lysozymes from yeast. It was found that the activities of both the mutant lysozymes were higher than those of the wild-type lysozyme based on the results of the activity measurements against M. luteus (lytic activity) and glycol chitin. Moreover, 3D structures of K33N and K33A mutant lysozyme were solved by X-ray crystallographic analyses. The side chain of K33 in the wild-type lysozyme hydrogen bonded with N37 involved in the substrate-binding region, and the orientation of the side chain of N37 in K33 mutant lysozymes were different in the wild-type lysozyme. These results suggest that the enhancement of activity in K33N mutant lysozyme was due to an alteration in the orientation of the side chain of N37. On the other hand, K33N lysozyme was less stable than the wild-type lysozyme. Lysozyme may sacrifice its enzyme activity to acquire the conformational stability at position 33.
Goto
Takashi
T
Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582, Japan.
Ohkuri
Takatoshi
T
Shioi
Seijiro
S
Abe
Yoshito
Y
Imoto
Taiji
T
Ueda
Tadashi
T
D000818:Animals; D002645:Chickens; D018360:Crystallography, X-Ray; D004529:Egg White / Q000737:chemistry*; D004795:Enzyme Stability; D005260:Female; D008239:Lysine / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009113:Muramidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016296:Mutagenesis; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary
D009113:Muramidase; D008239:Lysine