Structure, mechanistic action, and essential residues of a GH-64 enzyme, laminaripentaose-producing beta-1,3-glucanase.
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 A 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 A, 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 (Thr(156), 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.
Wu
Hsin-Mao
HM
Department of Life Science, Institute of Molecular and Cellular Biology, National Tsing Hua University, Hsinchu 300, Taiwan.
Liu
Sheng-Wen
SW
Hsu
Ming-Tsung
MT
Hung
Chiu-Lien
CL
Lai
Chun-Chieh
CC
Cheng
Wen-Chi
WC
Wang
Hung-Jung
HJ
Li
Yaw-Kuen
YK
Wang
Wen-Ching
WC
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D003460:Crystallization; D018360:Crystallography, X-Ray; D043326:Glucan 1,3-beta-Glucosidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity
D001426:Bacterial Proteins; D009844:Oligosaccharides; D043326:Glucan 1,3-beta-Glucosidase