Endo-fucoidan hydrolases from glycoside hydrolase family 107 (GH107) display structural and mechanistic similarities to α-l-fucosidases from GH29.

Fucoidans are chemically complex and highly heterogeneous sulfated marine fucans from brown macro algae. Possessing a variety of physicochemical and biological activities, fucoidans are used as gelling and thickening agents in the food industry and have anticoagulant, antiviral, antitumor, antibacterial, and immune activities. Although fucoidan-depolymerizing enzymes have been identified, the molecular basis of their activity on these chemically complex polysaccharides remains largely uninvestigated. In this study, we focused on three glycoside hydrolase family 107 (GH107) enzymes: MfFcnA and two newly identified members, P5AFcnA and P19DFcnA, from a bacterial species of the genus Psychromonas Using carbohydrate-PAGE, we show that P5AFcnA and P19DFcnA are active on fucoidans that differ from those depolymerized by MfFcnA, revealing differential substrate specificity within the GH107 family. Using a combination of X-ray crystallography and NMR analyses, we further show that GH107 family enzymes share features of their structures and catalytic mechanisms with GH29 α-l-fucosidases. However, we found that GH107 enzymes have the distinction of utilizing a histidine side chain as the proposed acid/base catalyst in its retaining mechanism. Further interpretation of the structural data indicated that the active-site architectures within this family are highly variable, likely reflecting the specificity of GH107 enzymes for different fucoidan substructures. Together, these findings begin to illuminate the molecular details underpinning the biological processing of fucoidans.

Directed Evolution of Clostridium thermocellum ß-Glucosidase A Towards Enhanced Thermostability.

ß-Glucosidases are key enzymes in the process of cellulose utilization. It is the last enzyme in the cellulose hydrolysis chain, which converts cellobiose to glucose. Since cellobiose is known to have a feedback inhibitory effect on a variety of cellulases, ß-glucosidase can prevent this inhibition by hydrolyzing cellobiose to non-inhibitory glucose. While the optimal temperature of the Clostridium thermocellum cellulosome is 70 °C, C. thermocellum ß-glucosidase A is almost inactive at such high temperatures. Thus, in the current study, a random mutagenesis directed evolutionary approach was conducted to produce a thermostable mutant with Kcat and Km, similar to those of the wild-type enzyme. The resultant mutant contained two mutations, A17S and K268N, but only the former was found to affect thermostability, whereby the inflection temperature (Ti) was increased by 6.4 °C. A17 is located near the central cavity of the native enzyme. Interestingly, multiple alignments revealed that position 17 is relatively conserved, whereby alanine is replaced only by serine. Upon the addition of the thermostable mutant to the C. thermocellum secretome for subsequent hydrolysis of microcrystalline cellulose at 70 °C, a higher soluble glucose yield (243%) was obtained compared to the activity of the secretome supplemented with the wild-type enzyme.

Atypical cohesin-dockerin complex responsible for cell surface attachment of cellulosomal components: binding fidelity, promiscuity, and structural buttresses.

The rumen bacterium Ruminococcus flavefaciens produces a highly organized multienzyme cellulosome complex that plays a key role in the degradation of plant cell wall polysaccharides, notably cellulose. The R. flavefaciens cellulosomal system is anchored to the bacterial cell wall through a relatively small ScaE scaffoldin subunit, which bears a single type IIIe cohesin responsible for the attachment of two major dockerin-containing scaffoldin proteins, ScaB and the cellulose-binding protein CttA. Although ScaB recruits the catalytic machinery onto the complex, CttA mediates attachment of the bacterial substrate via its two putative carbohydrate-binding modules. In an effort to understand the structural basis for assembly and cell surface attachment of the cellulosome in R. flavefaciens, we determined the crystal structure of the high affinity complex (Kd = 20.83 nM) between the cohesin module of ScaE (CohE) and its cognate X-dockerin (XDoc) modular dyad from CttA at 1.97-Å resolution. The structure reveals an atypical calcium-binding loop containing a 13-residue insert. The results further pinpoint two charged specificity-related residues on the surface of the cohesin module that are responsible for specific versus promiscuous cross-strain binding of the dockerin module. In addition, a combined functional role for the three enigmatic dockerin inserts was established whereby these extraneous segments serve as structural buttresses that reinforce the stalklike conformation of the X-module, thus segregating its tethered complement of cellulosomal components from the cell surface. The novel structure of the RfCohE-XDoc complex sheds light on divergent dockerin structure and function and provides insight into the specificity features of the type IIIe cohesin-dockerin interaction.

Functional association of catalytic and ancillary modules dictates enzymatic activity in glycoside hydrolase family 43 ß-xylosidase.

ß-Xylosidases are hemicellulases that hydrolyze short xylo-oligosaccharides into xylose units, thus complementing endoxylanase degradation of the hemicellulose component of lignocellulosic substrates. Here, we describe the cloning, characterization, and kinetic analysis of a glycoside hydrolase family 43 ß-xylosidase (Xyl43A) from the aerobic cellulolytic bacterium, Thermobifida fusca. Temperature and pH optima of 55-60 °C and 5.5-6, respectively, were determined. The apparent K(m) value was 0.55 mM, using p-nitrophenyl xylopyranoside as substrate, and the catalytic constant (k(cat)) was 6.72 s(-1). T. fusca Xyl43A contains a catalytic module at the N terminus and an ancillary module (termed herein as Module-A) of undefined function at the C terminus. We expressed the two recombinant modules independently in Escherichia coli and examined their remaining catalytic activity and binding properties. The separation of the two Xyl43A modules caused the complete loss of enzymatic activity, whereas potent binding to xylan was fully maintained in the catalytic module and partially in the ancillary Module-A. Nondenaturing gel electrophoresis revealed a specific noncovalent coupling of the two modules, thereby restoring enzymatic activity to 66.7% (relative to the wild-type enzyme). Module-A contributes a phenylalanine residue that functions as an essential part of the active site, and the two juxtaposed modules function as a single functional entity.

Crystallization and preliminary X-ray characterization of a type III cohesin-dockerin complex from the cellulosome system of Ruminococcus flavefaciens.

In Ruminococcus flavefaciens, a predominant fibre-degrading bacterium found in ruminants, cellulosomal proteins are anchored to the bacterial cell wall through a relatively small ScaE scaffoldin which includes a single type III cohesin. The cotton-binding protein CttA consists of two cellulose-binding modules and a C-terminal modular pair (XDoc) comprising an X-module and a contiguous dockerin, which exhibits high affinity towards the ScaE cohesin. Seleno-L-methionine-labelled derivatives of the ScaE cohesin module and the XDoc from CttA have been expressed, copurified and cocrystallized. The crystals belonged to the tetragonal space group P4(3)2(1)2, with unit-cell parameters a = b = 78.7, c = 203.4 Å, and the unit cell contains a single cohesin-XDoc complex in the asymmetric unit. The diffraction data were phased to 2.0 Å resolution using the anomalous signal of the Se atoms.

Cohesin diversity revealed by the crystal structure of the anchoring cohesin from Ruminococcus flavefaciens.

The cellulosome is an intriguing multienzyme complex found in cellulolytic bacteria that plays a key role in the degradation of plant cell-wall polysaccharides. In Ruminococcus flavefaciens, a predominant fiber-degrading bacterium found in ruminants, the cellulosome is anchored to the bacterial cell wall through a relatively short ScaE scaffoldin. Determination of the crystal structure of the lone type-III ScaE cohesin from R. flavefaciens (Rf-CohE) was initiated as a part of a structural effort to define cellulosome assembly. The structure was determined at 1.95 A resolution by single-wavelength anomalous diffraction. This is the first detailed description of a crystal structure for a type-III cohesin, and its features were compared with those of the known type-I and type-II cohesin structures. The Rf-CohE module folds into a nine-stranded beta-sandwich with jellyroll topology, typically observed for cohesins, and includes two beta-flaps in the midst of beta-strands 4 and 8, similar to the type-II cohesin structures. However, the presence in Rf-CohE of an additional 13-residue alpha-helix located between beta-strands 8 and 9 represents a dramatic divergence from other known cohesin structures. The prominent alpha-helix is enveloped by an extensive N-terminal loop, not observed in any other known cohesin, which embraces the helix presumably enhancing its stability. A planar surface at the upper portion of the front face of the molecule, bordered by beta-flap 8, exhibits plausible dimensions and exposed amino acid residues to accommodate the dockerin-binding site.

Insights into the κ/ι-carrageenan metabolism pathway of some marine Pseudoalteromonas species.

Pseudoalteromonas is a globally distributed marine-associated genus that can be found in a broad range of aquatic environments, including in association with macroalgal surfaces where they may take advantage of these rich sources of polysaccharides. The metabolic systems that confer the ability to metabolize this abundant form of photosynthetically fixed carbon, however, are not yet fully understood. Through genomics, transcriptomics, microbiology, and specific structure-function studies of pathway components we address the capacity of newly isolated marine pseudoalteromonads to metabolize the red algal galactan carrageenan. The results reveal that the κ/ι-carrageenan specific polysaccharide utilization locus (CarPUL) enables isolates possessing this locus the ability to grow on this substrate. Biochemical and structural analysis of the enzymatic components of the CarPUL promoted the development of a detailed model of the κ/ι-carrageenan metabolic pathway deployed by pseudoalteromonads, thus furthering our understanding of how these microbes have adapted to a unique environmental niche.

Preliminary X-ray characterization of a novel type of anchoring cohesin from the cellulosome of Ruminococcus flavefaciens.

Ruminococcus flavefaciens is an anaerobic bacterium that resides in the gastrointestinal tract of ruminants. It produces a highly organized multi-enzyme cellulosome complex that plays a key role in the degradation of plant cell walls. ScaE is one of the critical structural components of its cellulosome that serves to anchor the complex to the cell wall. The seleno-L-methionine-labelled derivative of the ScaE cohesin module has been cloned, expressed, purified and crystallized. The crystals belong to space group C2, with unit-cell parameters a = 155.6, b = 69.3, c = 93.0 A, beta = 123.4 degrees, and contain four molecules in the asymmetric unit. Diffraction data were phased to 1.95 A using the anomalous signal from the Se atoms.

Crystallization and preliminary X-ray analysis of Acetivibrio cellulolyticus cellulosomal type II cohesin module: two versions having different linker lengths.

The second type II cohesin module of the cellulosomal scaffoldin polypeptide ScaB from Acetivibrio cellulolyticus (CohB2) was cloned into two constructs: one containing a short (five-residue) C-terminal linker (CohB2_S) and the second incorporating the full native 45-residue linker (CohB2_L). Both constructs encode proteins that also include the full native six-residue N-terminal linker. The CohB2_S and CohB2_L proteins were expressed, purified and crystallized in the orthorhombic crystal system, but with different unit cells and symmetries: space group P2(1)2(1)2(1) with unit-cell parameters a = 90.36, b = 68.65, c = 111.29 A for CohB2_S and space group P2(1)2(1)2 with unit-cell parameters a = 68.76, b = 159.22, c = 44.21 A for CohB2_L. The crystals diffracted to 2.0 and 2.9 A resolution, respectively. The asymmetric unit of CohB2_S contains three cohesin molecules, while that of CohB2_L contains two molecules.

Characterization of Calflagin, a Flagellar Calcium-Binding Protein from Trypanosoma congolense.

BACKGROUND: Identification of species-specific trypanosome molecules is important for laboratory- and field-based research into epidemiology and disease diagnosis. Although Trypanosoma congolense is the most important trypanosome pathogen of cattle in Africa, no species-specific molecules found in infective bloodstream forms (BSF) of the parasites have been identified, thus limiting development of diagnostic tests. METHODS: Immuno-mass spectrometric methods were used to identify a protein that is recognized by a T. congolense-specific monoclonal antibody (mAb) Tc6/42.6.4. The identified molecule was expressed as a recombinant protein in E. coli and was tested in several immunoassays for its ability to interact with the mAb. The three dimensional structure of the protein was modeled and compared to crystal- and NMR-structures of the homologous proteins from T. cruzi and T. brucei respectively, in order to examine structural differences leading to the different immunoreactivity of the T. congolense molecule. Enzyme-linked immunosorbent assays (ELISA) were used to measure antibodies produced by trypanosome-infected African cattle in order to assess the potential for use of T. congolense calflagin in a serodiagnostic assay. RESULTS: The antigen recognized by the T. congolense-specific mAb Tc6/42.6.4 was identified as a flagellar calcium-binding protein, calflagin. The recombinant molecule showed immunoreactivity with the T. congolense-specific mAb confirming that it is the cognate antigen. Immunofluorescence experiments revealed that Ca2+ modulated the localization of the calflagin molecule in trypanosomes. Structural modelling and comparison with calflagin homologues from other trypanosomatids revealed four non-conserved regions on the surface of the T. congolense molecule that due to differences in surface chemistry and structural topography may form species-specific epitopes. ELISAs using the recombinant calflagin as antigen to detect antibodies in trypanosome-infected cattle showed that the majority of cattle had antibody responses. Area under the Receiver-Operating Characteristic (ROC) curves, associated with host IgG and IgM, were calculated to be 0.623 and 0.709 respectively, indicating a positive correlation between trypanosome infection and the presence of anti-calflagin antibodies. CONCLUSIONS: While calflagin is conserved among different species of African trypanosomes, our results show that T. congolense calflagin possesses unique epitopes that differentiate this protein from homologues in other trypanosome species. MAb Tc6/42.6.4 has clear utility as a laboratory tool for identifying T. congolense. T. congolense calflagin has potential as a serodiagnostic antigen and should be explored further for its utility in antigen-detection assays for diagnosis of cattle infections.

Standalone cohesin as a molecular shuttle in cellulosome assembly.

The cellulolytic bacterium Ruminococcus flavefaciens of the herbivore rumen produces an elaborate cellulosome system, anchored to the bacterial cell wall via the covalently bound scaffoldin ScaE. Dockerin-bearing scaffoldins also bind to an autonomous cohesin of unknown function, called cohesin G (CohG). Here, we demonstrate that CohG binds to the scaffoldin-borne dockerin in opposite orientation on a distinct site, relative to that of ScaE. Based on these structural data, we propose that the complexed dockerin is still available to bind ScaE on the cell surface. CohG may thus serve as a molecular shuttle for delivery of scaffoldins to the bacterial cell surface.

Intermodular linker flexibility revealed from crystal structures of adjacent cellulosomal cohesins of Acetivibrio cellulolyticus.

Cellulosome complexes comprise an intercalated set of multimodular dockerin-containing enzymatic subunits connected to cohesin-containing nonenzymatic subunits called scaffoldins. The adjoining modules in each cellulosomal subunit are interconnected by a variety of linker segments of different lengths and composition. The exact role of the cellulosomal linkers has yet to be described, although it is assumed that they contribute to the architecture and action of the cellulosome by providing the protein subunits with flexibility and by providing spacers between the enzymatic modules that could enhance interactions with the cellulose substrate. Here we present four crystal structures of Acetivibrio cellulolyticus cellulosomal type II cohesins with linker extensions. Two of the structures represent two different crystal forms (trigonal and orthorhombic) of the same N-terminal cohesin module (CohB1) together with its full (6-residue) native C-terminal linker, derived from scaffoldin B. The other two structures belong to the adjacent (second) cohesin module (CohB2), each of which was crystallized with the same 6-residue linker segment, but now positioned at the N-terminus and with either a truncated (5-residue) or a full-length (45-residue) C-terminal linker, respectively. Comparison between the two CohB1 structures revealed significant differences in the conformation of their equivalent C-terminal linker segment. In one crystal form a helical conformation was observed, as opposed to an extended conformation in the other. The CohB2 structures also displayed diverse conformations in their linker segments. In these structures, different linker conformations were observed in the individual molecules within the asymmetric unit of each structure. This conformational diversity implies that the linkers may adopt alternative conformations in their natural environment, consistent with varying environmental conditions. The findings suggest that linkers can play an important role in the assembly, dynamics and function of the cellulosomal components.