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

Abstract

Laminaripentaose (L5)‐producing β‐1,3‐glucanases can preferentially cleave the triple‐helix curdlan into β‐1,3‐glucooligosaccharides, especially L5. In this study, a newly identified member of the glycoside hydrolase family 64, β‐1,3‐glucanase from Streptomyces pratensis (SpGlu64A), was functionally and structurally characterized. SpGlu64A shared highest identity (30%) with a β‐1,3‐glucanase from Streptomyces matensis. The purified SpGlu64A showed maximal activity at pH 7.5 and 50 °C, and exhibited strict substrate specificity toward curdlan (83.1 U·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 (α/β) 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 β‐1,3‐glucan. A catalytic framework (β6–β9–β10) 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 β‐1,3‐glucanases but will also provide a basis for further enzyme engineering.