Shabir Najmudin

Xyloglucan Is Recognized by Carbohydrate-binding Modules That Interact with β-Glucan Chains

Enzyme systems that attack the plant cell wall contain noncatalytic carbohydrate-binding modules (CBMs) that mediate attachment to this composite structure and play a pivotal role in maximizing the hydrolytic process. Although xyloglucan, which includes a backbone of β-1,4-glucan decorated primarily with xylose residues, is a key component of the plant cell wall, CBMs that bind to this polymer have not been identified. Here we showed that the C-terminal domain of the modular Clostridium thermocellum enzyme CtCel9D-Cel44A (formerly known as CelJ) comprises a novel CBM (designated CBM44) that binds with equal affinity to cellulose and xyloglucan. We also showed that accommodation of xyloglucan side chains is a general feature of CBMs that bind to single cellulose chains. The crystal structures of CBM44 and the other CBM (CBM30) in CtCel9D-Cel44A display a β-sandwich fold. The concave face of both CBMs contains a hydrophobic platform comprising three tryptophan residues that can accommodate up to five glucose residues. The orientation of these aromatic residues is such that the bound ligand would adopt the twisted conformation displayed by cello-oligosaccharides in solution. Mutagenesis studies confirmed that the hydrophobic platform located on the concave face of both CBMs mediates ligand recognition. In contrast to other CBMs that bind to single polysaccharide chains, the polar residues in the binding cleft of CBM44 play only a minor role in ligand recognition. The mechanism by which these proteins are able to recognize linear and decorated β-1,4-glucans is discussed based on the structures of CBM44 and the other CBMs that bind single cellulose chains.

Putting an N-Terminal End to the Clostridium Thermocellum Xylanase Xyn10B Story: Crystal Structure of the Cbm22-1-Gh10 Modules Complexed with Xylohexaose.

In general, plant cell wall degrading enzymes are modular proteins containing catalytic domains linked to one or more non-catalytic carbohydrate-binding modules (CBMs). Xyn10B from Clostridium thermocellum is a typical modular enzyme containing an N-terminal family 22 CBM (CBM22-1), a family 10 glycoside hydrolase catalytic domain (GH10), a second CBM22 (CBM22-2), a dockerin sequence and a C-terminal family 1 carbohydrate esterase (CE1) catalytic domain. The structure of the N-terminal bi-modular CBM22-1-GH10 component of Xyn10B has been determined using a SeMet derivative by SAD to 2.5Å. The data was extended to 2.0Å for the non-SeMet mutant complexed with xylohexaose. CBM22-1-GH10 is a 60kDa protein with an E337A mutation to render the GH10 subunit inactive. Three of the six xylose residues of xylohexaose are shown to be bound in the inactivated GH10 substrate binding cleft, with the other three sugars presumably disordered in the solvent channel. The protein is a dimer in the asymmetric unit with extensive surface contacts between the two GH10 modules and between the CBM22-1 and GH10 modules. Residues from helix H4 of the GH10 module provide the major contacts by fitting into the minor groove of the CBM22-1 module. The orientation of CBM22-1 is such that it would allow the substrate to be loosely bound and subsequently delivered to the active site in a processive manner.

Structure of the bovine eye lens protein γB(γII)-crystallin at 1.47 Å

The molecular structure of calf γB-crystallin (previously called γII), a lens-specific protein, has been refined to a crystallographic R factor of 18.1% for all reflection data, between 8.0 and 1.47 A, 25 959 hkl measured at 293 (1) K. 230 water molecules have been defined by difference Fourier techniques and included in a restrained least-squares refinement. Difference Fourier maps clearly indicated the presence of multiple sites for the sulfur atoms of Cys 18 and Cys 22 which were therefore given coupled second-site occupancies during the refinement. The sulfur atom in the major position of Cys 22 is in the reduced state. Either of the Cys 18 sites can form a high-energy disulfide bridge with the minor position of Cys 22. The position of the carboxy terminus and many other surface side chains have been further defined including the RGD signal peptide. The hydration of the backbone and the interdomain region has been analysed. 27 water molecules make extensive contacts to a single protein molecule and thus contribute to its stability.

Purification, crystallization and preliminary X-ray crystallographic studies on acetolactate decarboxylase.

Acetolactate decarboxylase has the unique ability to decarboxylate both enantiomers of acetolactate to give a single enantiomer of the decarboxylation product, (R)-acetoin. A gene coding for alpha-acetolactate decarboxylase from Bacillus brevis (ATCC 11031) was cloned and overexpressed in B. subtilis. The enzyme was purified in two steps to homogeneity prior to crystallization. Three different diffraction-quality crystal forms were obtained by the hanging-drop vapour-diffusion method using a number of screening conditions. The best crystal form is suitable for structural studies and was grown from solutions containing 20% PEG 2000 MME, 10 mM cadmium chloride and 0.1 M Tris-HCl pH 7.0. They grew to a maximum dimension of approximately 0.4 mm and belong to the trigonal space group P3(1,2)21, with unit-cell parameters a = 47.0, c = 198.9 A. A complete data set was collected to 2 A from a single native crystal using synchrotron radiation.

Understanding How Noncatalytic Carbohydrate Binding Modules Can Display Specificity for Xyloglucan

Background: Carbohydrate binding modules (CBMs) contribute to the enzymatic degradation of complex polysaccharide structures. Results: New CBMs display specificity for decorated glucans through an extensive hydrophobic platform that interacts with both backbone and side chain structures. Conclusion: CBMs that bind to complex β-glucans exploit different components of these ligands as specificity determinants. Significance: CBMs can utilize the side chains of decorated glucans as specificity determinants. Plant biomass is central to the carbon cycle and to environmentally sustainable industries exemplified by the biofuel sector. Plant cell wall degrading enzymes generally contain noncatalytic carbohydrate binding modules (CBMs) that fulfil a targeting function, which enhances catalysis. CBMs that bind β-glucan chains often display broad specificity recognizing β1,4-glucans (cellulose), β1,3-β1,4-mixed linked glucans and xyloglucan, a β1,4-glucan decorated with α1,6-xylose residues, by targeting structures common to the three polysaccharides. Thus, CBMs that recognize xyloglucan target the β1,4-glucan backbone and only accommodate the xylose decorations. Here we show that two closely related CBMs, CBM65A and CBM65B, derived from EcCel5A, a Eubacterium cellulosolvens endoglucanase, bind to a range of β-glucans but, uniquely, display significant preference for xyloglucan. The structures of the two CBMs reveal a β-sandwich fold. The ligand binding site comprises the β-sheet that forms the concave surface of the proteins. Binding to the backbone chains of β-glucans is mediated primarily by five aromatic residues that also make hydrophobic interactions with the xylose side chains of xyloglucan, conferring the distinctive specificity of the CBMs for the decorated polysaccharide. Significantly, and in contrast to other CBMs that recognize β-glucans, CBM65A utilizes different polar residues to bind cellulose and mixed linked glucans. Thus, Gln106 is central to cellulose recognition, but is not required for binding to mixed linked glucans. This report reveals the mechanism by which β-glucan-specific CBMs can distinguish between linear and mixed linked glucans, and show how these CBMs can exploit an extensive hydrophobic platform to target the side chains of decorated β-glucans.

Novel Clostridium thermocellum Type I Cohesin-Dockerin Complexes Reveal a Single Binding Mode

Background: In general, dockerins present two homologous cohesin-binding interfaces, which confer increased flexibility into cellulosomes. Results: The structure of two novel Coh-Doc complexes reveals a dockerin single-binding mode. Conclusion: Single-binding mode dockerins bind, preferentially, to cell surface cohesins. Significance: The dual binding mode is a property of cellulosomal dockerins. Protein-protein interactions play a pivotal role in a large number of biological processes exemplified by the assembly of the cellulosome. Integration of cellulosomal components occurs through the binding of type I cohesin modules located in a non-catalytic molecular scaffold to type I dockerin modules located at the C terminus of cellulosomal enzymes. The majority of type I dockerins display internal symmetry reflected by the presence of two essentially identical cohesin-binding surfaces. Here we report the crystal structures of two novel Clostridium thermocellum type I cohesin-dockerin complexes (CohOlpC-Doc124A and CohOlpA-Doc918). The data revealed that the two dockerins, Doc918 and Doc124A, are unusual because they lack the structural symmetry required to support a dual binding mode. Thus, in both cases, cohesin recognition is dominated by residues located at positions 11, 12, and 19 of one of the dockerin binding surfaces. The alternative binding mode is not possible (Doc918) or highly limited (Doc124A) because residues that assume the critical interacting positions, when dockerins are reoriented by 180°, make steric clashes with the cohesin. In common with a third dockerin (Doc258) that also presents a single binding mode, Doc124A directs the appended cellulase, Cel124A, to the surface of C. thermocellum and not to cellulosomes because it binds preferentially to type I cohesins located at the cell envelope. Although there are a few exceptions, such as Doc918 described here, these data suggest that there is considerable selective pressure for the evolution of a dual binding mode in type I dockerins that direct enzymes into cellulosomes.

Family 46 Carbohydrate-binding Modules Contribute to the Enzymatic Hydrolysis of Xyloglucan and β-1,3–1,4-Glucans through Distinct Mechanisms

Background: CBMs are, generally, functionally and structurally autonomous from their associated catalytic domains. Results: The structure of a novel cellulase, BhCel5B, reveals that the appended carbohydrate-binding module, CBM46, extends the enzyme catalytic cleft. Conclusion: CBM46 targets BhCel5B to xyloglucan and is part of the catalytic cleft required for the hydrolysis of β-1,3–1,4-glucans. Significance: CBM46 has a dual role in the hydrolysis of complex carbohydrates by BhCel5B. Structural carbohydrates comprise an extraordinary source of energy that remains poorly utilized by the biofuel sector as enzymes have restricted access to their substrates within the intricacy of plant cell walls. Carbohydrate active enzymes (CAZYmes) that target recalcitrant polysaccharides are modular enzymes containing noncatalytic carbohydrate-binding modules (CBMs) that direct enzymes to their cognate substrate, thus potentiating catalysis. In general, CBMs are functionally and structurally autonomous from their associated catalytic domains from which they are separated through flexible linker sequences. Here, we show that a C-terminal CBM46 derived from BhCel5B, a Bacillus halodurans endoglucanase, does not interact with β-glucans independently but, uniquely, acts cooperatively with the catalytic domain of the enzyme in substrate recognition. The structure of BhCBM46 revealed a β-sandwich fold that abuts onto the region of the substrate binding cleft upstream of the active site. BhCBM46 as a discrete entity is unable to bind to β-glucans. Removal of BhCBM46 from BhCel5B, however, abrogates binding to β-1,3–1,4-glucans while substantially decreasing the affinity for decorated β-1,4-glucan homopolymers such as xyloglucan. The CBM46 was shown to contribute to xyloglucan hydrolysis only in the context of intact plant cell walls, but it potentiates enzymatic activity against purified β-1,3–1,4-glucans in solution or within the cell wall. This report reveals the mechanism by which a CBM can promote enzyme activity through direct interaction with the substrate or by targeting regions of the plant cell wall where the target glucan is abundant.

Heterodimeric nitrate reductase (NapAB) from Cupriavidus necator H16: purification, crystallization and preliminary X-ray analysis

The periplasmic nitrate reductase from Cupriavidus necator (also known as Ralstonia eutropha) is a heterodimer that is able to reduce nitrate to nitrite. It comprises a 91 kDa catalytic subunit (NapA) and a 17 kDa subunit (NapB) that is involved in electron transfer. The larger subunit contains a molybdenum active site with a bis-molybdopterin guanine dinucleotide cofactor as well as one [4Fe-4S] cluster, while the small subunit is a di-haem c-type cytochrome. Crystals of the oxidized form of this enzyme were obtained using polyethylene glycol 3350 as precipitant. A single crystal grown at the High Throughput Crystallization Laboratory of the EMBL in Grenoble diffracted to beyond 1.5 A at the ESRF (ID14-1), which is the highest resolution reported to date for a nitrate reductase. The unit-cell parameters are a = 142.2, b = 82.4, c = 96.8 A, beta = 100.7 degrees, space group C2, and one heterodimer is present per asymmetric unit.

Complexity of the Ruminococcus flavefaciens cellulosome reflects an expansion in glycan recognition

Significance Plant cell wall (PCW) polysaccharide degradation is an important biological and industrial process. Noncatalytic carbohydrate binding modules (CBMs) fulfill a critical targeting function in PCW depolymerization. Ruminococcus flavefaciens synthesizes a highly efficient PCW degrading apparatus. Here, six previously unidentified R. flavefaciens CBM families were identified that targeted β-glucans, β-mannans, and pectins. Crystal structures of these CBMs revealed that recognition of β-glucans and β-mannans was mediated by differences in the conformation of conserved aromatic residues in the ligand binding cleft. A cluster of basic residues in CBM77 confers calcium-independent recognition of homogalacturonan. This report shows that the expansion of protein modules in the cellulosome of R. flavefaciens contributes to an extended CBM profile that supports efficient PCW degradation. The breakdown of plant cell wall (PCW) glycans is an important biological and industrial process. Noncatalytic carbohydrate binding modules (CBMs) fulfill a critical targeting function in PCW depolymerization. Defining the portfolio of CBMs, the CBMome, of a PCW degrading system is central to understanding the mechanisms by which microbes depolymerize their target substrates. Ruminococcus flavefaciens, a major PCW degrading bacterium, assembles its catalytic apparatus into a large multienzyme complex, the cellulosome. Significantly, bioinformatic analyses of the R. flavefaciens cellulosome failed to identify a CBM predicted to bind to crystalline cellulose, a key feature of the CBMome of other PCW degrading systems. Here, high throughput screening of 177 protein modules of unknown function was used to determine the complete CBMome of R. flavefaciens. The data identified six previously unidentified CBM families that targeted β-glucans, β-mannans, and the pectic polysaccharide homogalacturonan. The crystal structures of four CBMs, in conjunction with site-directed mutagenesis, provide insight into the mechanism of ligand recognition. In the CBMs that recognize β-glucans and β-mannans, differences in the conformation of conserved aromatic residues had a significant impact on the topology of the ligand binding cleft and thus ligand specificity. A cluster of basic residues in CBM77 confers calcium-independent recognition of homogalacturonan, indicating that the carboxylates of galacturonic acid are key specificity determinants. This report shows that the extended repertoire of proteins in the cellulosome of R. flavefaciens contributes to an extended CBMome that supports efficient PCW degradation in the absence of CBMs that specifically target crystalline cellulose.

Cell-surface Attachment of Bacterial Multienzyme Complexes Involves Highly Dynamic Protein-Protein Anchors

Background: Cohesin-dockerin interactions support the binding of multienzyme complexes (cellulosomes) onto the cell surface. Results: The structures of novel Coh-Doc complexes reveal a dual binding mode. Conclusion: A dual binding mode is present in Coh-Doc interactions supporting cell-surface attachment. Significance: The dual binding mode introduces flexibility into highly populated multienzyme complexes attached to the cell surface. Protein-protein interactions play a pivotal role in the assembly of the cellulosome, one of natures most intricate nanomachines dedicated to the depolymerization of complex carbohydrates. The integration of cellulosomal components usually occurs through the binding of type I dockerin modules located at the C terminus of the enzymes to cohesin modules located in the primary scaffoldin subunit. Cellulosomes are typically recruited to the cell surface via type II cohesin-dockerin interactions established between primary and cell-surface anchoring scaffoldin subunits. In contrast with type II interactions, type I dockerins usually display a dual binding mode that may allow increased conformational flexibility during cellulosome assembly. Acetivibrio cellulolyticus produces a highly complex cellulosome comprising an unusual adaptor scaffoldin, ScaB, which mediates the interaction between the primary scaffoldin, ScaA, through type II cohesin-dockerin interactions and the anchoring scaffoldin, ScaC, via type I cohesin-dockerin interactions. Here, we report the crystal structure of the type I ScaB dockerin in complex with a type I ScaC cohesin in two distinct orientations. The data show that the ScaB dockerin displays structural symmetry, reflected by the presence of two essentially identical binding surfaces. The complex interface is more extensive than those observed in other type I complexes, which results in an ultra-high affinity interaction (Ka ∼1012 m). A subset of ScaB dockerin residues was also identified as modulating the specificity of type I cohesin-dockerin interactions in A. cellulolyticus. This report reveals that recruitment of cellulosomes onto the cell surface may involve dockerins presenting a dual binding mode to incorporate additional flexibility into the quaternary structure of highly populated multienzyme complexes.