Structure, mechanistic action, and essential residues of a GH-64 enzyme, laminaripentaose-producing β-1,3-glucanase

Abstract

Laminaripentaose-producing β-1,3-glucanase (LPHase), a member of glycoside hydrolase family 64, cleaves a longchain polysaccharide β-1,3-glucan into specific pentasaccharide oligomers. The crystal structure of LPHase from Streptomyces matensis DIC-108 was solved to 1.62 Å 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 (α/β) domain, forming a wide-open groove between the two domains. The liganded crystal structure was also solved to 1.80 Å, 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 (Glu154 and Asp170) and four other sugar-binding residues (Thr156, Asn158, Trp163, and Thr167). Molecular modeling using a laminarihexaose as a substrate suggests roles for Glu154 and Asp170 as acid and base catalysts, respectively, whereas the side chains of Thr156, Asn158, and Trp163 demarcate subsite +5. Site-directed mutagenesis of Glu154 and Asp170 confirms that both carboxylates are essential for catalysis. Together, our results suggest that LPHase uses a direct displacement mechanism involving Glu154 and Asp170 to cleave a β-1,3-glucan into specific α-pentasaccharide oligomers. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.