Structural and Kinetic Dissection of the endo‐α‐1, 2‐Mannanase Activity of Bacterial GH99 Glycoside Hydrolases from Bacteroides spp. Issue 5 (8th December 2014)

• Record Type: Journal Article
• Authors: Hakki, Zalihe; Thompson, Andrew J.; Bellmaine, Stephanie; Speciale, Gaetano; Davies, Gideon J.; Williams, Spencer J.
• Is Part Of: Chemistry. Volume 21:Issue 5(2015)
• Page Start: 1966
• Page End: 1977
• Publication Date: 2014-12-08
• Subjects: Chemistry -- Periodicals; 540
• DOI: 10.1002/chem.201405539
• Languages: English
• ISSNs: 0947-6539

Abstract

<abstract abstract-type="main" xml:lang="en"> <title>Abstract</title> <p>Glycoside hydrolase family 99 (GH99) was created to categorize sequence‐related glycosidases possessing <italic>endo</italic>‐α‐mannosidase activity: the cleavage of mannosidic linkages within eukaryotic N‐glycan precursors (Glc<sub>1–3</sub>Man<sub>9</sub>GlcNAc<sub>2</sub>), releasing mono‐, di‐ and triglucosylated‐mannose (Glc<sub>1–3</sub>‐1, 3‐Man). GH99 family members have recently been implicated in the ability of <italic>Bacteroides</italic> spp., present within the gut microbiota, to metabolize fungal cell wall α‐mannans, releasing α‐1, 3‐mannobiose by hydrolysing αMan‐1, 3‐αMan→1, 2‐αMan‐1, 2‐αMan sequences within branches off the main α‐1, 6‐mannan backbone. We report the development of a series of substrates and inhibitors, which we use to kinetically and structurally characterise this novel <italic>endo</italic>‐α‐1, 2‐mannanase activity of bacterial GH99 enzymes from <italic>Bacteroides thetaiotaomicron</italic> and <italic>xylanisolvens</italic>. These data reveal an approximate 5 kJ mol<sup>−1</sup> preference for mannose‐configured substrates in the −2 subsite (relative to glucose), which inspired the development of a new inhibitor, α‐mannopyranosyl‐1, 3‐isofagomine (ManIFG), the most potent (bacterial) GH99 inhibitor reported to date. X‐ray structures of ManIFG or a substrate in complex with wild‐type or inactive mutants, respectively, of <italic>B. xylanisolvens</italic> GH99 reveal the structural basis for binding to <sc>D</sc>‐mannose‐ rather than <sc>D</sc>‐glucose‐configured substrates.</p> </abstract>