Two high-resolution structures of potato endo-1,3-β-glucanase reveal subdomain flexibility with implications for substrate binding

Endo-1,3-beta-glucanases are widely distributed among bacteria, fungi and higher plants. They are responsible for hydrolysis of the glycosidic bond in specific polysaccharides with tracts of unsubstituted beta-1,3-linked glucosyl residues. The plant enzymes belong to glycoside hydrolase family 17 (GH17) and are also members of class 2 of pathogenesis-related (PR) proteins. X-ray diffraction data were collected to 1.40 and 1.26 angstrom resolution from two crystals of endo-1,3-beta-glucanase from Solanum tuberosum (potato, cultivar Desiree) which, despite having a similar packing framework, represented two separate crystal forms. In particular, they differed in the Matthews coefficient and are consequently referred to as higher density (HD; 1.40 angstrom resolution) and lower density (LD; 1.26 angstrom resolution) forms. The general fold of the protein resembles that of other known plant endo-1,3-beta-glucanases and is defined by a (beta/alpha)(8)-barrel with an additional subdomain built around the C-terminal half of the barrel. The structures revealed high flexibility of the subdomain, which forms part of the catalytic cleft. Comparison with structures of other GH17 endo-1,3-beta-glucanases revealed differences in the arrangement of the secondary-structure elements in this region, which can be correlated with sequence variability and may suggest distinct substrate-binding patterns. The crystal structures revealed an unusual packing mode, clearly visible in the LD structure, caused by the presence of the C-terminal His(6) tag, which extends from the compact fold of the enzyme molecule and docks in the catalytic cleft of a neighbouring molecule. In this way, an infinite chain of His-tag-linked protein molecules is formed along the c direction.