Mutational analysis of amino acid residues involved in catalytic activity of a family 18 chitinase from tulip bulbs.
We expressed chitinase-1 (TBC-1) from tulip bulbs (Tulipa bakeri) in E. coli cells and used site-directed mutagenesis to identify amino acid residues essential for catalytic activity. Mutations at Glu-125 and Trp-251 completely abolished enzyme activity, and activity decreased with mutations at Asp-123 and Trp-172 when glycolchitin was the substrate. Activity changed with the mutations of Trp-251 to one of several amino acids with side-chains of little hydrophobicity, suggesting that hydrophobic interaction of Trp-251 is important for the activity. Molecular dynamics (MD) simulation analysis with hevamine as the model compound showed that the distance between Asp-123 and Glu-125 was extended by mutation of Trp-251. Kinetic studies of Trp-251-mutated chitinases confirmed these various phenomena. The results suggested that Glu-125 and Trp-251 are essential for enzyme activity and that Trp-251 had a direct role in ligand binding.
Laboratories of Protein Chemistry and Engineering, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan.
Keisuke
K
Suzukawa
Takeshi
T
Yamagami
Takayuki
T
Ohnuma
Hideki
H
Hirakawa
Satoru
S
Kuhara
Yoichi
Y
Aso
Masatsune
M
Ishiguro
D019943:Amino Acid Substitution; D000596:Amino Acids / Q000032:analysis* / Q000235:genetics*; D001665:Binding Sites; D002384:Catalysis; D002686:Chitin / Q000031:analogs & derivatives* / Q000378:metabolism; D002688:Chitinases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D003198:Computer Simulation; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000378:metabolism; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D008958:Models, Molecular; D009113:Muramidase / Q000737:chemistry / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010940:Plant Proteins; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D031447:Tulipa / Q000201:enzymology* / Q000235:genetics
D000596:Amino Acids; D017931:DNA Primers; D010940:Plant Proteins; D011994:Recombinant Proteins; D002686:Chitin; C031610:glycolchitin; D002688:Chitinases; C063764:hevamine; D009113:Muramidase