5dgr

Publication Abstract from PubMed

Exo-beta-D-glucosaminidase (EC 3.2.1.165) from Photobacterium profundum (PpGlcNase) is an inverting glycoside hydrolase (GH) belonging to family 9. We have determined the three-dimensional structure of PpGlcNase to describe the first structure-function relationship of an exo-type GH9 glycosidase. PpGlcNase has a narrow and straight active site pocket, in contrast to the long glycan binding cleft of a GH9 endoglucanase. This is because PpGlcNase has a long loop, which blocks the position corresponding to subsites -4 to -2 of the endoglucanase. The pocket shape of PpGlcNase explains its substrate preference for a beta1,4-linkage at the non-reducing terminus. D139, D143 and E555 in the active site were located near the beta-O1 hydroxyl group of GlcN, with D139 and D143 holding a nucleophilic water molecule for hydrolysis. The D139A, D143A and E555A mutants significantly decreased hydrolytic activity, indicating their essential role. Of these mutants, D139A exclusively exhibited glycosynthase activity using alpha-GlcN-F and GlcN as substrates, to produce (GlcN)2. Using saturation mutagenesis at D139, we obtained D139E as the best glycosynthase. Compared with the wild type, the hydrolytic activity of D139E was significantly suppressed (< 0.1%) whereas the fluoride ion-releasing activity also decreased (< 3%). Therefore, the glycosynthase activity of D139E was lower than that of glycosynthases previously created from other inverting GHs. Mutation at the nucleophilic water holder is a general strategy for creating an effective glycosynthase from inverting GHs. However, for GH9, where two acidic residues seem to share the catalytic base role, mutation of D139 might inevitably reduce fluoride ion-releasing activity.

The crystal structure of an inverting glycoside hydrolase family 9 exo-beta-D-glucosaminidase and the design of glycosynthase.,Honda Y, Arai S, Suzuki K, Kitaoka M, Fushinobu S Biochem J. 2015 Nov 30. pii: BJ20150966. PMID:26621872^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.