Nicola Smith

Evidence for a dual binding mode of dockerin modules to cohesins

The assembly of proteins that display complementary activities into macromolecular complexes is critical to cellular function. One such enzyme complex, of environmental significance, is the plant cell wall degrading apparatus of anaerobic bacteria, termed the cellulosome. The complex assembles through the interaction of enzyme-derived “type I dockerin” modules with the multiple “cohesin” modules of the scaffolding protein. Clostridium thermocellum type I dockerin modules contain a duplicated 22-residue sequence that comprises helix-1 and helix-3, respectively. The crystal structure of a C. thermocellum type I cohesin-dockerin complex showed that cohesin recognition was predominantly through helix-3 of the dockerin. The sequence duplication is reflected in near-perfect 2-fold structural symmetry, suggesting that both repeats could interact with cohesins by a common mechanism in wild-type (WT) proteins. Here, a helix-3 disrupted mutant dockerin is used to visualize the reverse binding in which the dockerin mutant is indeed rotated 180° relative to the WT dockerin such that helix-1 now dominates recognition of its protein partner. The dual binding mode is predicted to impart significant plasticity into the orientation of the catalytic subunits within this supramolecular assembly, which reflects the challenges presented by the degradation of a heterogeneous, recalcitrant, insoluble substrate by a tethered macromolecular complex.

The Location of the Ligand-binding Site of Carbohydrate-binding Modules That Have Evolved from a Common Sequence Is Not Conserved

Polysaccharide-degrading enzymes are generally modular proteins that contain non-catalytic carbohydrate-binding modules (CBMs), which potentiate the activity of the catalytic module. CBMs have been grouped into sequence-based families, and three-dimensional structural data are available for half of these families. Clostridium thermocellum xylanase 11A is a modular enzyme that contains a CBM from family 6 (CBM6), for which no structural data are available. We have determined the crystal structure of this module to a resolution of 2.1 Å. The protein is a β-sandwich that contains two potential ligand-binding clefts designated cleft A and B. The CBM interacts primarily with xylan, and NMR spectroscopy coupled with site-directed mutagenesis identified cleft A, containing Trp-92, Tyr-34, and Asn-120, as the ligand-binding site. The overall fold of CBM6 is similar to proteins in CBM families 4 and 22, although surprisingly the ligand-binding site in CBM4 and CBM22 is equivalent to cleft B in CBM6. These structural data define a superfamily of CBMs, comprising CBM4, CBM6, and CBM22, and demonstrate that, although CBMs have evolved from a relatively small number of ancestors, the structural elements involved in ligand recognition have been assembled at different locations on the ancestral scaffold.

Structural and biochemical evidence for a boat-like transition state in |[beta]|-mannosidases

Enzyme inhibition through mimicry of the transition state is a major area for the design of new therapeutic agents. Emerging evidence suggests that many retaining glycosidases that are active on alpha- or beta-mannosides harness unusual B2,5 (boat) transition states. Here we present the analysis of 25 putative beta-mannosidase inhibitors, whose Ki values range from nanomolar to millimolar, on the Bacteroides thetaiotaomicron beta-mannosidase BtMan2A. B2,5 or closely related conformations were observed for all tightly binding compounds. Subsequent linear free energy relationships that correlate log Ki with log Km/kcat for a series of active center variants highlight aryl-substituted mannoimidazoles as powerful transition state mimics in which the binding energy of the aryl group enhances both binding and the degree of transition state mimicry. Support for a B2,5 transition state during enzymatic beta-mannosidase hydrolysis should also facilitate the design and exploitation of transition state mimics for the inhibition of retaining alpha-mannosidases--an area that is emerging for anticancer therapeutics.

Mechanistic insights into a Ca2+-dependent family of α-mannosidases in a human gut symbiont

Colonic bacteria, exemplified by Bacteroides thetaiotaomicron, play a key role in maintaining human health by harnessing large families of glycoside hydrolases (GHs) to exploit dietary polysaccharides and host glycans as nutrients. Such GH family expansion is exemplified by the 23 family GH92 glycosidases encoded by the B. thetaiotaomicron genome. Here we show that these are alpha-mannosidases that act via a single displacement mechanism to utilize host N-glycans. The three-dimensional structure of two GH92 mannosidases defines a family of two-domain proteins in which the catalytic center is located at the domain interface, providing acid (glutamate) and base (aspartate) assistance to hydrolysis in a Ca(2+)-dependent manner. The three-dimensional structures of the GH92s in complex with inhibitors provide insight into the specificity, mechanism and conformational itinerary of catalysis. Ca(2+) plays a key catalytic role in helping distort the mannoside away from its ground-state (4)C(1) chair conformation toward the transition state.

Understanding How Diverse β-Mannanases Recognize Heterogeneous Substrates

The mechanism by which polysaccharide-hydrolyzing enzymes manifest specificity toward heterogeneous substrates, in which the sequence of sugars is variable, is unclear. An excellent example of such heterogeneity is provided by the plant structural polysaccharide glucomannan, which comprises a backbone of beta-1,4-linked glucose and mannose units. beta-Mannanases, located in glycoside hydrolase (GH) families 5 and 26, hydrolyze glucomannan by cleaving the glycosidic bond of mannosides at the -1 subsite. The mechanism by which these enzymes select for glucose or mannose at distal subsites, which is critical to defining their substrate specificity on heterogeneous polymers, is currently unclear. Here we report the biochemical properties and crystal structures of both a GH5 mannanase and a GH26 mannanase and describe the contributions to substrate specificity in these enzymes. The GH5 enzyme, BaMan5A, derived from Bacillus agaradhaerens, can accommodate glucose or mannose at both its -2 and +1 subsites, while the GH26 Bacillus subtilis mannanase, BsMan26A, displays tight specificity for mannose at its negative binding sites. The crystal structure of BaMan5A reveals that a polar residue at the -2 subsite can make productive contact with the substrate 2-OH group in either its axial (as in mannose) or its equatorial (as in glucose) configuration, while other distal subsites do not exploit the 2-OH group as a specificity determinant. Thus, BaMan5A is able to hydrolyze glucomannan in which the sequence of glucose and mannose is highly variable. The crystal structure of BsMan26A in light of previous studies on the Cellvibrio japonicus GH26 mannanases CjMan26A and CjMan26C reveals that the tighter mannose recognition at the -2 subsite is mediated by polar interactions with the axial 2-OH group of a (4)C(1) ground state mannoside. Mutagenesis studies showed that variants of CjMan26A, from which these polar residues had been removed, do not distinguish between Man and Glc at the -2 subsite, while one of these residues, Arg 361, confers the elevated activity displayed by the enzyme against mannooligosaccharides. The biological rationale for the variable recognition of Man- and Glc-configured sugars by beta-mannanases is discussed.

Mannose Foraging by Bacteroides thetaiotaomicron STRUCTURE AND SPECIFICITY OF THE β-MANNOSIDASE, BtMan2A

The human colonic bacterium Bacteroides thetaiotaomicron, which plays an important role in maintaining human health, produces an extensive array of exo-acting glycoside hydrolases (GH), including 32 family GH2 glycoside hydrolases. Although it is likely that these enzymes enable the organism to utilize dietary and host glycans as major nutrient sources, the biochemical properties of these GH2 glycoside hydrolases are currently unclear. Here we report the biochemical properties and crystal structure of the GH2 B. thetaiotaomicron enzyme BtMan2A. Kinetic analysis demonstrates that BtMan2A is a β-mannosidase in which substrate binding energy is provided principally by the glycone binding site, whereas aglycone recognition is highly plastic. The three-dimensional structure, determined to a resolution of 1.7Å, reveals a five-domain structure that is globally similar to the Escherichia coli LacZ β-galactosidase. The catalytic center is housed mainly within a (β/α)8 barrel although the N-terminal domain also contributes to the active site topology. The nature of the substrate-binding residues is quite distinct from other GH2 enzymes of known structure, instead they are similar to other clan GH-A enzymes specific for manno-configured substrates. Mutagenesis studies, informed by the crystal structure, identified a WDW motif in the N-terminal domain that makes a significant contribution to catalytic activity. The observation that this motif is invariant in GH2 mannosidases points to a generic role for these residues in this enzyme class. The identification of GH-A clan and GH2 specific residues in the active site of BtMan2A explains why this enzyme is able to harness substrate binding at the proximal glycone binding site more efficiently than mannan-hydrolyzing glycoside hydrolases in related enzyme families. The catalytic properties of BtMan2A are consistent with the flexible nutrient acquisition displayed by the colonic bacterium.

Crystal structures of a family 8 polysaccharide lyase reveal open and highly occluded substrate-binding cleft conformations

Bacterial enzymatic degradation of glycosaminoglycans such as hyaluronan and chondroitin is facilitated by polysaccharide lyases. Family 8 polysaccharide lyase (PL8) enzymes contain at least two domains: one predominantly composed of α‐helices, the α‐domain, and another predominantly composed of β‐sheets, the β‐domain. Simulation flexibility analyses indicate that processive exolytic cleavage of hyaluronan, by PL8 hyaluronate lyases, is likely to involve an interdomain shift, resulting in the opening/closing of the substrate‐binding cleft between the α‐ and β‐domains, facilitating substrate translocation. Here, the Streptomyces coelicolor A3(2) PL8 enzyme was recombinantly expressed in and purified from Escherichia coli and biochemically characterized as a hyaluronate lyase. By using X‐ray crystallography its structure was solved in complex with hyaluronan and chondroitin disaccharides. These findings show key catalytic interactions made by the different substrates, and on comparison with all other PL8 structures reveals that the substrate‐binding cleft of the S. coelicolor enzyme is highly occluded. A third structure of the enzyme, harboring a mutation of the catalytic tyrosine, created via site‐directed mutagenesis, interestingly revealed an interdomain shift that resulted in the opening of the substrate‐binding cleft. These results add further support to the proposed processive mechanism of action of PL8 hyaluronate lyases and may indicate that the mechanism of action is likely to be universally used by PL8 hyaluronate lyases. Proteins 2011.

Structures of Two Truncated Phage-Tail Hyaluronate Lyases from Streptococcus Pyogenes Serotype M1.

The crystal structures of truncated forms of the Streptococcus pyogenes phage-encoded hyaluronate lyases HylP2 and HylP3 were determined by molecular replacement to 1.6 and 1.9 A resolution, respectively. The truncated forms crystallized in a hexagonal space group, forming a trimer around the threefold crystallographic axis. The arrangement of the fold is very similar to that observed in the structure of the related hyaluronate lyase HylP1. The structural elements putatively involved in substrate recognition are found to be conserved in both the HylP2 and HylP3 fragments.

The gene encoding the ribulose monophosphate pathway enzyme, 3-hexulose-6-phosphate synthase, from Aminomonas aminovorus C2A1 is adjacent to coding sequences that exhibit similarity to histidine biosynthesis enzymes.

In an attempt to understand better the organisation of genes encoding enzymes of the ribulose monophosphate pathway (RuMP), the 3-hexulose 6-phosphate synthase gene (hps) and flanking sequences were cloned from the obligate methylotroph Aminomonas aminovorus C2A1. To date only three hps containing gene clusters from methylotrophs have been characterised and these contain genes encoding other RuMP enzymes. However, hps from A. aminovorus C2A1 was shown to be adjacent to coding sequences for products with sequence similarity to histidine biosynthesis enzymes. Furthermore, none of the hps homologue containing gene clusters, from genome sequences previously analysed or analysed in this paper, were similar in organisation to that of A. aminovorus C2A1.

Paediatric musculoskeletal matters (pmm) – collaborative development of an online evidence based interactive learning tool and information resource for education in paediatric musculoskeletal medicine

BackgroundWe describe the collaborative development of an evidence based, free online resource namely ‘paediatric musculoskeletal matters’ (pmm). This resource was developed with the aim of reaching a wide range of health professionals to increase awareness, knowledge and skills within paediatric musculoskeletal medicine, thereby facilitating early diagnosis and referral to specialist care.MethodsEngagement with stakeholder groups (primary care, paediatrics, musculoskeletal specialties and medical students) informed the essential ‘core’ learning outcomes to derive content of pmm. Representatives from stakeholder groups, social science and web development experts transformed the learning outcomes into a suitable framework. Target audience representatives reviewed the framework and their opinion was gathered using an online survey (n = 74) and focus groups (n = 2). Experts in paediatric musculoskeletal medicine peer reviewed the content and design.ResultsUser preferences informed design with mobile, tablet and web compatible versions to facilitate access, various media and formats to engage users and the content presented in module format (i.e. Clinical assessment, Investigations and management, Limping child, Joint pain by site, Swollen joint(s) and Resources).ConclusionsWe propose that our collaborative and evidence-based approach has ensured that pmm is user-friendly, with readily accessible, suitable content, and will help to improve access to paediatric musculoskeletal medicine education. The content is evidence-based with the design and functionality of pmm to facilitate optimal and ‘real life’ access to information. pmm is targeted at medical students and the primary care environment although messages are transferable to all health care professionals involved in the care of children and young people.