Structural and functional analysis of SpGlu64A: a novel glycoside hydrolase family 64 laminaripentaose-producing β-1,3-glucanase from Streptomyces pratensis

Laminaripentaose (L5)-producing beta-1,3-glucanases can preferentially cleave the triple-helix curdlan into beta-1,3-glucooligosaccharides, especially L5. In this study, a newly identified member of the glycoside hydrolase family 64, beta-1,3-glucanase from Streptomyces pratensis (SpGlu64A), was functionally and structurally characterized. SpGlu64A shared highest identity (30%) with a beta-1,3-glucanase from Streptomyces matensis. The purified SpGlu64A showed maximal activity at pH 7.5 and 50 C-degrees, and exhibited strict substrate specificity toward curdlan (83.1 U center dot mg(-1)). It efficiently hydrolyzed curdlan to produce L5 as the end product. The overall structure of SpGlu64A consisted of a barrel domain and a mixed (alpha/beta) domain, which formed an unusually wide groove with a crescent-like structure. In the two complex structures (SpGlu64A-L3 and SpGlu64A-L4), two oligosaccharide chains were captured and the triple-helical structure was relatively compatible with the wide groove, which suggested the possibility of binding to the triple-helical beta-1,3-glucan. A catalytic framework (beta 6-beta 9-beta 10) and the steric hindrance formed by the side chains of residues Y161, N163, and H393 in the catalytic groove were predicted to complete the exotype-like cleavage manner. On the basis of the structure, a fusion protein with the CBM56 domain (SpGlu64A-CBM) and a mutant (Y161F; by site-directed mutation) were obtained, with 1.2- and 1.7-fold increases in specific activity, respectively. Moreover, the combined expression of SpGlu64A-CBM and -Y161F improved the enzyme activity by 2.63-fold. The study will not only be helpful in understanding the reaction mechanism of beta-1,3-glucanases but will also provide a basis for further enzyme engineering.