title: Major change in regiospecificity for the exo-1,3-β-glucanase from Candida albicans following its conversion to a glycosynthase.
abstract: The exo-1,3-β-glucanase (Exg) from Candida albicans is involved in cell wall β-d-glucan metabolism and morphogenesis through its hydrolase and transglycosidase activities. Previous work has shown that both these activities strongly favor β-1,3-linkages. The E292S Exg variant displayed modest glycosynthase activity using α-d-glucopyranosyl fluoride (α-GlcF) as the donor and pNP-β-d-glucopyranoside (pNPGlc) as the acceptor but surprisingly showed a marked preference for synthesizing β-1,6-linked over β-1,3- and β-1,4-linked disaccharide products. With pNPXyl as the acceptor, the preference became β-1,4 over β-1,3. The crystal structure of the glycosynthase bound to both of its substrates, α-GlcF and pNPGlc, is the first such ternary complex structure to be determined. The results revealed that the donor bound in the -1 subsite, as expected, while the acceptor was oriented in the +1 subsite to facilitate β-1,6-linkage, thereby supporting the results from solution studies. A second crystal structure containing the major product of glycosynthesis, pNP-gentiobiose, showed that the -1 subsite allows another docking position for the terminal sugar; i.e., one position is set up for catalysis, whereas the other is an intermediate stage prior to the displacement of water from the active site by the incoming sugar hydroxyls. The +1 subsite, an aromatic "clamp", permits several different sugar positions and orientations, including a 180° flip that explains the observed variable regiospecificity. The p-nitrophenyl group on the acceptor most likely influences the unexpectedly observed β-1,6-specificity through its interaction with F229. These results demonstrate that tailoring the specificity of a particular glycosynthase depends not only on the chemical structure of the acceptor but also on understanding the structural basis of the promiscuity of the native enzyme.
authors.affiliation: Biochemistry Department, University of Otago , P.O. Box 56, Dunedin 9054, New Zealand.
authors.forename: Y
authors.initials: Y
authors.lastname: Nakatani
authors.forename: D S
authors.initials: DS
authors.lastname: Larsen
authors.forename: S M
authors.initials: SM
authors.lastname: Cutfield
authors.forename: J F
authors.initials: JF
authors.lastname: Cutfield
mesh_terms: D002176:Candida albicans / Q000201:enzymology*; D018360:Crystallography, X-Ray; D005656:Fungal Proteins / Q000737:chemistry* / Q000378:metabolism; D043326:Glucan 1,3-beta-Glucosidase / Q000737:chemistry* / Q000378:metabolism; D006006:Glycogen Synthase / Q000737:chemistry* / Q000378:metabolism; D017433:Protein Structure, Secondary; D013379:Substrate Specificity / Q000502:physiology
chemical_list: D005656:Fungal Proteins; D006006:Glycogen Synthase; D043326:Glucan 1,3-beta-Glucosidase