Research Article: Bacteroides thetaiotaomicron generates diverse α-mannosidase activities through subtle evolution of a distal substrate-binding motif

Date Published: May 01, 2018

Publisher: International Union of Crystallography

Author(s): Andrew J. Thompson, Richard J. Spears, Yanping Zhu, Michael D. L. Suits, Spencer J. Williams, Harry J. Gilbert, Gideon J. Davies.


Analysing two sequence-related bacterial glycoside hydrolase family 92 mannosidases with distinct functions, a structural basis for their varied specificities is revealed.

Partial Text

Prokaryote-encoded α-mannosidase enzymes, particularly those deployed by the human gut microbiota, have garnered significant interest in recent years owing to their importance in digestive health. Furthermore, these highly accessible model proteins share significant structural and mechanistic similarity with complex mammalian homologues, and have provided new insights into the intricate nucleophilic substitution reactions that are the hallmark of mannosidase chemistry (Suits et al., 2010 ▸; Thompson et al., 2012 ▸; Offen et al., 2009 ▸; Tailford et al., 2008 ▸). Emerging research has revealed that certain symbiotic gut-resident bacteria maintain a selective advantage over microbial competitors through the ability to catabolize α-linked mannopolysaccharides as a sole carbon source (Cuskin et al., 2015 ▸). One such highly prevalent bacterium, Bacteroides thetaiotaomicron (Bt), encodes large numbers of enzymes from α-mannoside-specific glycoside hydrolase (GH) families, including GH38, GH76, GH92 and GH125 [family definitions according to the sequence-based CAZy classification system (Lombard et al., 2014 ▸); see and]. Within the Bt genome, enzymes from these families are organized and regulated in a highly systematic fashion within polysaccharide-utilization loci (PULs; see Martens et al., 2009 ▸), which allows the rapid and simultaneous expression of a complete molecular ‘toolkit’ to catalyse the breakdown of otherwise inaccessible high-mannose dietary components such as yeast α-mannan and mammalian high-mannose N-glycans. Unlike many other carbohydrate-hydrolysing pathways, which allow shared access to metabolic byproducts in a symbiotic relationship with the host, Bt uniquely employs mannan/mannoside-targeted PULs in a purely ‘selfish’ mechanism (Cuskin et al., 2015 ▸), emphasizing a selectively advantageous role for these gene/enzyme catalogues compared with fellow gut-resident microbial species. As such, the biochemical properties and correct regulation of enzymes within these loci is crucial in the maintenance of a balanced and diverse gut microbial environ­ment and in the overall healthy functioning of the microbiota.

The three-dimensional structures of BT3130 and BT3965 both reveal a two-domain enzyme that appears to be well conserved with published examples of GH92 α-mannosidases. These enzymes feature a pocket-like central cavity composed of structural elements from both the N-terminal and C-terminal domains (Zhu et al., 2010 ▸; Robb et al., 2017 ▸). Kinetic analysis of site-specific enzyme variants, together with ligand-complex structures, have shown that this pocket-like region comprises the catalytic active site (Zhu et al., 2010 ▸). Primary-sequence analysis and direct comparisons with available structures demonstrate strong conservation of key amino-acid side chains within this region of both BT3130 and BT3965, suggesting that the observed differences in substrate specificity are likely to be conferred by minor, local structural differences, while both the global fold and the overall reaction mechanism for these diverse α-mannosidases are broadly maintained (see Figs. 1 ▸ and 2 ▸). Indeed, quantitative structural comparison using the DALI server (Holm & Rosenström, 2010 ▸) shows that the three-dimensional structures of BT3130 and BT3965 have high similarity, with an r.m.s.d. of 1.4 Å mapped over 738 matched Cα positions (sequence identity = 40%, Z-score = 48.7).

The following references are cited in the Supporting Information for this article: Ashkenazy et al. (2016 ▸) and Landau et al. (2005 ▸).




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