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Title: The functional effect of Gly209 and Ile213 substitutions on lysozyme activity of family 19 chitinase encoded by cyanophage Ma-LMM01
Authors: Hosoda, Naohiko
Kurokawa, Yoichi
Sako, Yoshihiko  kyouindb  KAKEN_id
Nagasaki, Keizo
Yoshida, Takashi  kyouindb  KAKEN_id
Hiroishi, Shingo
Author's alias: 吉田, 天士
Keywords: Microcystis
Cyanophage
Family 19 chitinase
Site-directed mutagenesis
Issue Date: Jul-2011
Publisher: Springer Japan
Journal title: Fisheries Science
Volume: 77
Issue: 4
Start page: 665
End page: 670
Abstract: ORF69 in the cyanophage infecting Microcystis aeruginosa, Ma-LMM01, shows homology to the family 19 chitinases where the catalytic domain has structural similarity to lysozyme. Chitinases hydrolyze chitin, a β-1, 4-linked monopolymer of N-acetylglucosamine (GlcNAc); whereas lysozymes hydrolyzes peptidoglycan, alternating β-1, 4-linked copolymers of N-acetylmuramic acid (MurNAc) and GlcNAc. Using amino acid sequence comparison to ORF69, the putative sugar binding residues, Gln162 and Lys165, from the barley chitinase (the model enzyme for the family 19 chitinases) corresponding to subsites −4 and −3 were found to be replaced with Gly209 and Ile213, respectively, in ORF69. To analyze their contribution to substrate binding affinity, ORF69 was cloned into Escherichia coli; and two mutant proteins G209Q and I213K were prepared using site-directed mutagenesis. The wild-type gene product (gp69) showed both lysozyme and chitinase activities. In contrast, the I213K mutant showed a decrease (70%) in lysozyme activity and a significant increase (50%) in chitinase activity; whereas, the G209Q mutant almost completely abolished both enzyme activities. The data suggest the Ile213 residue is involved in recognizing the substrate MurNAc; and Gly209 has significant contribution in chitinase and lysozyme activities for the wild-type gp69.
Rights: The final publication is available at www.springerlink.com
This is not the published version. Please cite only the published version. この論文は出版社版でありません。引用の際には出版社版をご確認ご利用ください。
URI: http://hdl.handle.net/2433/143681
DOI(Published Version): 10.1007/s12562-011-0352-9
Appears in Collections:Journal Articles

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