Lookup NU author(s): Dr Natalie Ferry,
Professor Harry Gilbert
One of the most intriguing features of the 90 glycoside hydrolase families (GHs) is the range of specificities displayed by different members of the same family, whereas the catalytic apparatus and mechanism are often invariant. Family GH26 predominantly comprises beta-1,4 mannanases; however, a bifunctional Clostridium thermocellum GH26 member (hereafter CtLic26A) displays a markedly different specificity. We show that CtLic26A is a lichenase, specific for mixed (Glc beta 1,4Glc beta 1,4Glc beta 1,3)(n) oligo- and polysaccharides, and displays no activity on manno-configured substrates or beta-1,4-linked homopolymers of glucose or xylose. The three-dimensional structure of the native form of CtLic26A has been solved at 1.50-angstrom resolution, revealing a characteristic (beta/alpha)s barrel with Glu-109 and Glu-222 acting as the catalytic acid/base and nucleophile in a double-displacement mechanism. The complex with the competitive inhibitor, Glc-beta-1,3-isofagomine (K-i 1 mu M), at 1.60 angstrom sheds light on substrate recognition in the -2 and -1 subsites and illuminates why the enzyme is specific for lichenan-based substrates. Hydrolysis of beta-mannosides by GH26 members is thought to proceed through transition states in the B2+5 (boat) conformation in which structural distinction of glucosides versus mannosides reflects not the configuration at C2 but the recognition of the pseudoaxial O3 of the B2+5 conformation. We suggest a different conformational itinerary for the GH26 enzymes active on gluco-configured substrates.
Author(s): Taylor EJ, Goyal A, Guerreiro CIPD, Prates JAM, Money VA, Ferry N, Morland C, Planas A, Macdonald JA, Stick RV, Gilbert HJ, Fontes CMGA, Davies GJ
Publication type: Article
Journal: Journal of Biological Chemistry
ISSN (print): 0021-9258
ISSN (electronic): 1083-351X
Publisher: American Society for Biochemistry and Molecular Biology, Inc.
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