Structure and Kinetic Investigation of Streptococcus pyogenes Family GH38 α-Mannosidase

BACKGROUND: The enzymatic hydrolysis of alpha-mannosides is catalyzed by glycoside hydrolases (GH), termed alpha-mannosidases. These enzymes are found in different GH sequence-based families. Considerable research has probed the role of higher eukaryotic "GH38" alpha-mannosides that play a key role in the modification and diversification of hybrid N-glycans; processes with strong cellular links to cancer and autoimmune disease. The most extensively studied of these enzymes is the Drosophila GH38 alpha-mannosidase II, which has been shown to be a retaining alpha-mannosidase that targets both alpha-1,3 and alpha-1,6 mannosyl linkages, an activity that enables the enzyme to process GlcNAc(Man)(5)(GlcNAc)(2) hybrid N-glycans to GlcNAc(Man)(3)(GlcNAc)(2). Far less well understood is the observation that many bacterial species, predominantly but not exclusively pathogens and symbionts, also possess putative GH38 alpha-mannosidases whose activity and specificity is unknown. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that the Streptococcus pyogenes (M1 GAS SF370) GH38 enzyme (Spy1604; hereafter SpGH38) is an alpha-mannosidase with specificity for alpha-1,3 mannosidic linkages. The 3D X-ray structure of SpGH38, obtained in native form at 1.9 A resolution and in complex with the inhibitor swainsonine (K(i) 18 microM) at 2.6 A, reveals a canonical GH38 five-domain structure in which the catalytic "-1" subsite shows high similarity with the Drosophila enzyme, including the catalytic Zn(2+) ion. In contrast, the "leaving group" subsites of SpGH38 display considerable differences to the higher eukaryotic GH38s; features that contribute to their apparent specificity. CONCLUSIONS/SIGNIFICANCE: Although the in vivo function of this streptococcal GH38 alpha-mannosidase remains unknown, it is shown to be an alpha-mannosidase active on N-glycans. SpGH38 lies on an operon that also contains the GH84 hexosaminidase (Spy1600) and an additional putative glycosidase. The activity of SpGH38, together with its genomic context, strongly hints at a function in the degradation of host N- or possibly O-glycans. The absence of any classical signal peptide further suggests that SpGH38 may be intracellular, perhaps functioning in the subsequent degradation of extracellular host glycans following their initial digestion by secreted glycosidases

A serious game to improve engagement with web accessibility guidelines

This is an Accepted Manuscript of an article published by Taylor & Francis in Behaviour & Information Technology on 07 Jan 2020, available online: https://www.tandfonline.com/doi/full/10.1080/0144929X.2019.171145

Carbohydrate Recognition by an Architecturally Complex α-N-Acetylglucosaminidase from Clostridium perfringens

CpGH89 is a large multimodular enzyme produced by the human and animal pathogen Clostridium perfringens. The catalytic activity of this exo-α-d-N-acetylglucosaminidase is directed towards a rare carbohydrate motif, N-acetyl-β-d-glucosamine-α-1,4-d-galactose, which is displayed on the class III mucins deep within the gastric mucosa. In addition to the family 89 glycoside hydrolase catalytic module this enzyme has six modules that share sequence similarity to the family 32 carbohydrate-binding modules (CBM32s), suggesting the enzyme has considerable capacity to adhere to carbohydrates. Here we suggest that two of the modules, CBM32-1 and CBM32-6, are not functional as carbohydrate-binding modules (CBMs) and demonstrate that three of the CBMs, CBM32-3, CBM32-4, and CBM32-5, are indeed capable of binding carbohydrates. CBM32-3 and CBM32-4 have a novel binding specificity for N-acetyl-β-d-glucosamine-α-1,4-d-galactose, which thus complements the specificity of the catalytic module. The X-ray crystal structure of CBM32-4 in complex with this disaccharide reveals a mode of recognition that is based primarily on accommodation of the unique bent shape of this sugar. In contrast, as revealed by a series of X-ray crystal structures and quantitative binding studies, CBM32-5 displays the structural and functional features of galactose binding that is commonly associated with CBM family 32. The functional CBM32s that CpGH89 contains suggest the possibility for multivalent binding events and the partitioning of this enzyme to highly specific regions within the gastrointestinal tract

Human gut Bacteroidetes can utilize yeast mannan through a selfish mechanism

Yeasts, which have been a component of the human diet for at least 7,000 years, possess an elaborate cell wall α-mannan. The influence of yeast mannan on the ecology of the human microbiota is unknown. Here we show that yeast α-mannan is a viable food source for the Gram-negative bacterium Bacteroides thetaiotaomicron, a dominant member of the microbiota. Detailed biochemical analysis and targeted gene disruption studies support a model whereby limited cleavage of α-mannan on the surface generates large oligosaccharides that are subsequently depolymerized to mannose by the action of periplasmic enzymes. Co-culturing studies showed that metabolism of yeast mannan by B. thetaiotaomicron presents a ‘selfish’ model for the catabolism of this difficult to breakdown polysaccharide. Genomic comparison with B. thetaiotaomicron in conjunction with cell culture studies show that a cohort of highly successful members of the microbiota has evolved to consume sterically-restricted yeast glycans, an adaptation that may reflect the incorporation of eukaryotic microorganisms into the human diet

Structure of the Streptococcus pneumoniae surface protein and adhesin PfbA.

PfbA (plasmin- and fibronectin-binding protein A) is an extracellular Streptococcus pneumoniae cell-wall attached surface protein that binds to fibronectin, plasmin, and plasminogen. Here we present a structural analysis of the surface exposed domains of PfbA using a combined approach of X-ray crystallography and small-angle X-ray scattering (SAXS). The crystal structure of the PfbA core domain, here called PfbAβ, determined to 2.28 Å resolution revealed an elongated 12-stranded parallel β-helix fold, which structure-based comparisons reveal is most similar to proteins with carbohydrate modifying activity. A notable feature of the PfbAβ is an extensive cleft on one face of the protein with electrochemical and spatial features that are analogous to structurally similar carbohydrate-active enzymes utilizing this feature for substrate accommodation. Though this cleft displays a combination of basic amino acid residues and solvent exposed aromatic amino acids that are distinct features for recognition of carbohydrates, no obvious arrangement of amino acid side chains that would constitute catalytic machinery is evident. The pseudo-atomic SAXS model of a larger fragment of PfbA suggests that it has a relatively well-ordered structure with the N-terminal and core domains of PfbA adopting an extend organization and reveals a novel structural class of surface exposed pneumococcal matrix molecule adhesins

The Contribution of Structural Proteomics to Understanding the Function of Hypothetical Proteins

Le Conseil des ministres a adopté, le 20 avril 2008, la réforme des institutions que le Président Nicolas Sarkozy, avait annoncé, en juillet dernier, avec l’idée de «moderniser» la Cinquième République, «sans pour autant tourner la page». Ce texte prévoit d’accroître les prérogatives du Parlement (notamment, partage de la fixation de l’ordre du jour entre le gouvernement et le Parlement, limitation de l’usage de l’art. 49-3 qui permet de faire adopter un texte de loi sans vote…). La réforme d..

Schematic representation of the modular architecture of PfbA and structure of the parallel β-helix core domain.

<p>(A) The PfbA architecture include the FSIRK and LPXTG sequence motifs that are common Gram-positive export signal peptide and streptococcal cell-wall adherence features, respectively. The surface exposed α/β and cell-wall link domains are conserved across pneumococcal isolates. (B) Cartoon representation of the CAZyme-like 12-stranded β-helix domain solved by X-ray crystallography to 2.28 Å. The 90 residue (T139-Q230, coloured red) N-terminal initiator of the core domain is a conserved precursor of the parallel β-helix fold and includes the calcium-binding site established by the sidechains of E187 and H214 and the carbonyl oxygen of T225. A water coordinated by residues Q296, Q331, E333 and the carbonyl oxygen of G339 is reminiscent of the metal binding sites seen in polysaccharide lyases.</p

PfbAΔC pseudo-atomic solution model.

<p>(A) Experimental and theoretical SAXS data calculated for the (B) averaged <i>ab initio</i> surface representation of 10 independent DAMMIF calculations and (C), rigid body fit of the PfbAβ domain into the envelope. (D) I-TASSER generated model of the PfbA N-terminal α/β domain (residues 54–138). (E) CRYSOL calculated and experimental SAXS data for the (F) PfbAΔC composite structure generated from SAXS data with the program BUNCH. Individual models resulted in excellent fits to the data with χ_(DAMMIF) values in the range of 1.5–1.6.</p

PfbAβ surface features and putative carbohydrate accommodation Cleft.

<p>(A) Carbohydrate accommodation groove of <i>endo</i>-<i>N</i>-acetylglucosaminidase tailspike protein from the <i>E. coli</i> bacteriophage HK620 complexed with the substrate <i>O</i>-antigen hexasaccharide (orange). (B) A putative carbohydrate accommodation groove (grey) formed along the length of the PfbAβ domain has features conserved across carbohydrate processing factors. (C) A series of electropositive residues (blue) line this cleft (dashed line) which could interact with negatively charged polysaccharides. (D) The solvent accessible surface of PfbAβ colored by electrostatic potential also reveals the electropositive nature of the cleft and shows the branched nature of the cleft. (E) Phylogenetic mapping of homologous sequences to PfbA. Conserved residues are shown in purple and pink, neutral in white, ambiguous in yellow, and non-conserved in blue as per Consurf standard colouration scheme. (F) The electronegative residues Q296, Q331, E333 and carbonyl G339 coordinate a crystallographic water, are conserved at the base of the cleft branch point. This putative active centre is lined with the electropositive series of residues H293, K320, K327, K338, H377 and R405 which have features similar to carbohydate active lyases. This conserved region is circled in panel E.</p