Publication Abstract from PubMed
The exo-1,3-beta-glucanase (Exg) from Candida albicans is involved in cell wall beta-d-glucan metabolism and morphogenesis through its hydrolase and transglycosidase activities. Previous work has shown that both these activities strongly favor beta-1,3-linkages. The E292S Exg variant displayed modest glycosynthase activity using alpha-d-glucopyranosyl fluoride (alpha-GlcF) as the donor and pNP-beta-d-glucopyranoside (pNPGlc) as the acceptor but surprisingly showed a marked preference for synthesizing beta-1,6-linked over beta-1,3- and beta-1,4-linked disaccharide products. With pNPXyl as the acceptor, the preference became beta-1,4 over beta-1,3. The crystal structure of the glycosynthase bound to both of its substrates, alpha-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 beta-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 degrees flip that explains the observed variable regiospecificity. The p-nitrophenyl group on the acceptor most likely influences the unexpectedly observed beta-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.
Major Change in Regiospecificity for the Exo-1,3-beta-glucanase from Candida albicans following Its Conversion to a Glycosynthase.,Nakatani Y, Larsen DS, Cutfield SM, Cutfield JF Biochemistry. 2014 May 27;53(20):3318-26. doi: 10.1021/bi500239m. Epub 2014 May, 14. PMID:24804868[1]
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.
