標題: Characterization and identification of essential residues of the glycoside hydrolase family 64 laminaripentaose-producing--1, 3-glucanase
作者: Shrestha, Keshab Lal
Liu, Sheng-Wen
Huang, Chin-Ping
Wu, Hsin-Mao
Wang, Wen-Ching
Li, Yaw-Kuen
應用化學系
Department of Applied Chemistry
關鍵字: GH-64;Laminaripentaose-producing-1;3-glucanase;catalytic mechanism;essential residue;site-directed mutagenesis;chemical rescue
公開日期: 1-Aug-2011
摘要: Laminaripentaose-producing -1,3-glucanase (LPHase) from Streptomyces matensis DIC-108 uniquely catalyzes the hydrolysis of -1,3-glucan to release laminaripentaose as the predominant product. For studying this novel enzyme, the gene of LPHase was reconstructed with polymerase chain reaction and over-expressed in Escherichia coli. The recombinant wild-type enzyme and various mutants were further purified to 90 homogeneity on an ion-exchange chromatograph. The catalysis of the recombinant LPHase is confirmed to follow a one-step single-displacement mechanism with (1)H-NMR spectrometry. To determine the amino-acid residues essential for the catalysis, more than ten residues, including five highly conserved residuesAsp(143), Glu(154), Asp(170), Asp(376) and Asp(377), were mutated. Among the mutants, E154Q, E154G, D174N and D174G significantly lost catalytic activity. Further investigation with chemical rescue using sodium azide on E154G and D174G confirmed that Glu(154) functions as the general acid whereas Asp(170) serves as the general base in a catalytic turnover. This work is the first report that provides direct information for the identification of the essential residues of GH-64 through kinetic examination.
URI: http://dx.doi.org/10.1093/protein/gzr031
http://hdl.handle.net/11536/21127
ISSN: 1741-0126
DOI: 10.1093/protein/gzr031
期刊: PROTEIN ENGINEERING DESIGN & SELECTION
Volume: 24
Issue: 8
起始頁: 617
結束頁: 625
Appears in Collections:Articles


Files in This Item:

  1. 000292567400005.pdf