4M80

The structure of E292S glycosynthase variant of exo-1,3-beta-glucanase from Candida albicans at 1.85A resolution

Summary for 4M80

• Entry DOI: 10.2210/pdb4m80/pdb
• Related: 1CZ1 4M81 4M82
• Descriptor: EXO-1,3-BETA-GLUCANASE (2 entities in total)
• Functional Keywords: tim barrel, glycoside hydrolase family 5, glycoside hydrolase, cell wall hydrolase, glycosynthase, hydrolase
• Biological source: Candida albicans (yeast)
• Total number of polymer chains: 1
• Total formula weight: 45693.31

Authors

Nakatani, Y.,Cutfield, S.M.,Larsen, D.S.,Cutfield, J.F. (deposition date: 2013-08-12, release date: 2014-06-25, Last modification date: 2024-11-27)

Primary citation

Nakatani, Y.,Larsen, D.S.,Cutfield, S.M.,Cutfield, J.F.
Major Change in Regiospecificity for the Exo-1,3-beta-glucanase from Candida albicans following Its Conversion to a Glycosynthase.
Biochemistry, 53:3318-3326, 2014

PubMed 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.

PubMed: 24804868
DOI: 10.1021/bi500239m

Experimental method

X-RAY DIFFRACTION (1.858 Å)