標題: Structure, Mechanistic Action, and Essential Residues of a GH-64 Enzyme, Laminaripentaose-producing beta-1,3-Glucanase
作者: Wu, Hsin-Mao
Liu, Sheng-Wen
Hsu, Ming-Tsung
Hung, Chiu-Lien
Lai, Chun-Chieh
Cheng, Wen-Chi
Wang, Hung-Jung
Li, Yaw-Kuen
Wang, Wen-Ching
應用化學系
Department of Applied Chemistry
公開日期: 25-九月-2009
摘要: Laminaripentaose-producing beta-1,3-glucanase (LPHase), a member of glycoside hydrolase family 64, cleaves a long-chain polysaccharide beta-1,3-glucan into specific pentasaccharide oligomers. The crystal structure of LPHase from Streptomyces matensis DIC-108 was solved to 1.62 angstrom resolution using multiple-wavelength anomalous dispersion methods. The LPHase structure reveals a novel crescent-like fold; it consists of a barrel domain and a mixed (alpha/beta) domain, forming a wide-open groove between the two domains. The liganded crystal structure was also solved to 1.80 angstrom, showing limited conformational changes. Within the wide groove, a laminaritetraose molecule is found to sit in an electronegatively charged central region and is proximal to several conserved residues including two carboxylates (Glu(154) and Asp(170)) and four other sugar-binding residues (Thr156, Asn(158), Trp(163), and Thr(167)). Molecular modeling using a laminarihexaose as a substrate suggests roles for Glu(154) and Asp(170) as acid and base catalysts, respectively, whereas the side chains of Thr(156), Asn(158), and Trp(163) demarcate subsite +5. Site-directed mutagenesis of Glu(154) and Asp(170) confirms that both carboxylates are essential for catalysis. Together, our results suggest that LPHase uses a direct displacement mechanism involving Glu(154) and Asp(170) to cleave a beta-1,3-glucan into specific alpha-pentasaccharide oligomers.
URI: http://dx.doi.org/10.1074/jbc.M109.010983
http://hdl.handle.net/11536/6660
ISSN: 0021-9258
DOI: 10.1074/jbc.M109.010983
期刊: JOURNAL OF BIOLOGICAL CHEMISTRY
Volume: 284
Issue: 39
起始頁: 26708
結束頁: 26715
顯示於類別:期刊論文


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