Three-dimensional structure of a barley beta-D-glucan exohydrolase, a family 3 glycosyl hydrolase

Background: Cell walls of the starchy endosperm and young vegetative tissue s of barley (Hordeum vulgare) contain high levels of (1-->3, 1-->4)-beta-D- glucans. The (1-->3, 1-->4)-beta-D-glucans are hydrolysed during wall degra dation in germinated grain and during wall loosening in elongating coleopti les. These key processes of plant development are mediated by several polys accharide endohydrolases and exohydrolases. Results: The three-dimensional structure of barley beta-D-glucan exohydrola se isoenzyme Exol has been determined by X-ray crystallography. This is the first reported structure of a family 3 glycosyl hydrolase. The enzyme is a two-domain, globular protein of 605 amino acid residues and is N-glycosyla ted at three sites. The first 357 residues constitute an (alpha/beta)(8) TI M-barrel domain. The second domain consists of residues 374-559 arranged in a six-stranded beta sandwich, which contains a beta sheet of five parallel beta strands and one antiparallel beta strand, with three a helices on eit her side of the sheet. A glucose moiety is observed in a pocket at the inte rface of the two domains, where Asp285 and Glu491 are believed to be involv ed in catalysis. Conclusions: The pocket at the interface of the two domains is probably the active site of the enzyme. Because amino acid residues that line this acti ve-site pocket arise from both domains, activity could be regulated through the spatial disposition of the domains. Furthermore, there are sites on th e second domain that may bind carbohydrate, as suggested by previously publ ished kinetic data indicating that, in addition to the catalytic site, the enzyme has a second binding site specific for (1-->3,1-->4)-beta-D-glucans.