Mapping the deformability of natural and designed cellulosomes in solution.

BACKGROUND: Natural cellulosome multi-enzyme complexes, their components, and engineered 'designer cellulosomes' (DCs) promise an efficient means of breaking down cellulosic substrates into valuable biofuel products. Their broad uptake in biotechnology relies on boosting proximity-based synergy among the resident enzymes, but the modular architecture challenges structure determination and rational design. RESULTS: We used small angle X-ray scattering combined with molecular modeling to study the solution structure of cellulosomal components. These include three dockerin-bearing cellulases with distinct substrate specificities, original scaffoldins from the human gut bacterium Ruminococcus champanellensis (ScaA, ScaH and ScaK) and a trivalent cohesin-bearing designer scaffoldin (Scaf20L), followed by cellulosomal complexes comprising these components, and the nonavalent fully loaded Clostridium thermocellum CipA in complex with Cel8A from the same bacterium. The size analysis of Rg and Dmax values deduced from the scattering curves and corresponding molecular models highlight their variable aspects, depending on composition, size and spatial organization of the objects in solution. CONCLUSIONS: Our data quantifies variability of form and compactness of cellulosomal components in solution and confirms that this native plasticity may well be related to speciation with respect to the substrate that is targeted. By showing that scaffoldins or components display enhanced compactness compared to the free objects, we provide new routes to rationally enhance their stability and performance in their environment of action.

The laterally acquired GH5 ZgEngAGH5_4 from the marine bacterium Zobellia galactanivorans is dedicated to hemicellulose hydrolysis.

Cell walls of marine macroalgae are composed of diverse polysaccharides that provide abundant carbon sources for marine heterotrophic bacteria. Among them, Zobellia galactanivorans is considered as a model for studying algae-bacteria interactions. The degradation of typical algal polysaccharides, such as agars or alginate, has been intensively studied in this model bacterium, but the catabolism of plant-like polysaccharides is essentially uncharacterized. Here, we identify a polysaccharide utilization locus in the genome of Z. galactanivorans, induced by laminarin (ß-1,3-glucans), and containing a putative GH5 subfamily 4 (GH5_4) enzyme, currently annotated as a endoglucanase (ZgEngAGH5_4). A phylogenetic analysis indicates that ZgEngAGH5_4 was laterally acquired from an ancestral Actinobacteria We performed the biochemical and structural characterization of ZgEngAGH5_4 and demonstrated that this GH5 is, in fact, an endo-ß-glucanase, most active on mixed-linked glucan (MLG). Although ZgEngAGH5_4 and GH16 lichenases both hydrolyze MLG, these two types of enzymes release different series of oligosaccharides. Structural analyses of ZgEngAGH5_4 reveal that all the amino acid residues involved in the catalytic triad and in the negative glucose-binding subsites are conserved, when compared with the closest relative, the cellulase EngD from Clostridium cellulovorans, and some other GH5s. In contrast, the positive glucose-binding subsites of ZgEngAGH5_4 are different and this could explain the preference for MLG, with respect to cellulose or laminarin. Molecular dynamics computer simulations using different hexaoses reveal that the specificity for MLG occurs through the +1 and +2 subsites of the binding pocket that display the most important differences when compared with the structures of other GH5_4 enzymes.

X-ray Diffraction and Density Functional Theory Provide Insight into Vanadate Binding to Homohexameric Bromoperoxidase II and the Mechanism of Bromide Oxidation.

X-ray diffraction of native bromoperoxidase II (EC 1.11.1.18) from the brown alga Ascophyllum nodosum reveals at a resolution of 2.26 Å details of orthovanadate binding and homohexameric protein organization. Three dimers interwoven in contact regions and tightened by hydrogen-bond-clamped guanidinium stacks along with regularly aligned water molecules form the basic structure of the enyzme. Intra- and intermolecular disulfide bridges further stabilize the enzyme preventing altogether the protein from denaturing up to a temperature of 90 °C, as evident from dynamic light scattering and the on-gel ortho-dianisidine assay. Every monomer binds one equivalent of orthovanadate in a cavity formed from side chains of three histidines, two arginines, one lysine, serine, and tryptophan. Protein binding occurs primarily through hydrogen bridges and superimposed by Coulomb attraction according to thermochemical model on density functional level of theory (B3LYP/6-311++G**). The strongest attractor is the arginine side chain mimic N-methylguanidinium, enhancing in positive cooperative manner hydrogen bridges toward weaker acceptors, such as residues from lysine and serine. Activating hydrogen peroxide occurs in the thermochemical model by side-on binding in orthovanadium peroxoic acid, oxidizing bromide with virtually no activation energy to hydrogen bonded hypobromous acid.

Structure of the GH76 α-mannanase homolog, BT2949, from the gut symbiont Bacteroides thetaiotaomicron.

The large bowel microbiota, a complex ecosystem resident within the gastrointestinal tract of all human beings and large mammals, functions as an essential, nonsomatic metabolic organ, hydrolysing complex dietary polysaccharides and modulating the host immune system to adequately tolerate ingested antigens. A significant member of this community, Bacteroides thetaiotaomicron, has evolved a complex system for sensing and processing a wide variety of natural glycoproducts in such a way as to provide maximum benefit to itself, the wider microbial community and the host. The immense ability of B. thetaiotaomicron as a `glycan specialist' resides in its enormous array of carbohydrate-active enzymes, many of which are arranged into polysaccharide-utilization loci (PULs) that are able to degrade sugar polymers that are often inaccessible to other gut residents, notably α-mannan. The B. thetaiotaomicron genome encodes ten putative α-mannanases spread across various PULs; however, little is known about the activity of these enzymes or the wider implications of α-mannan metabolism for the health of both the microbiota and the host. In this study, SAD phasing of a selenomethionine derivative has been used to investigate the structure of one such B. thetaiotaomicron enzyme, BT2949, which belongs to the GH76 family of α-mannanases. BT2949 presents a classical (α/α)6-barrel structure comprising a large extended surface cleft common to other GH76 family members. Analysis of the structure in conjunction with sequence alignments reveals the likely location of the catalytic active site of this noncanonical GH76.

Structure and activity of the Streptococcus pyogenes family GH1 6-phospho-ß-glucosidase SPy1599.

The group A streptococcus Streptococcus pyogenes is the causative agent of a wide spectrum of invasive infections, including necrotizing fasciitis, scarlet fever and toxic shock syndrome. In the context of its carbohydrate chemistry, it is interesting that S. pyogenes (in this work strain M1 GAS SF370) displays a spectrum of oligosaccharide-processing enzymes that are located in close proximity on the genome but that the in vivo function of these proteins remains unknown. These proteins include different sugar transporters (SPy1593 and SPy1595), both GH125 α-1,6- and GH38 α-1,3-mannosidases (SPy1603 and SPy1604), a GH84 ß-hexosaminidase (SPy1600) and a putative GH2 ß-galactosidase (SPy1586), as well as SPy1599, a family GH1 `putative ß-glucosidase'. Here, the solution of the three-dimensional structure of SPy1599 in a number of crystal forms complicated by unusual crystallographic twinning is reported. The structure is a classical (ß/α)(8)-barrel, consistent with CAZy family GH1 and other members of the GH-A clan. SPy1599 has been annotated in sequence depositions as a ß-glucosidase (EC 3.2.1.21), but no such activity could be found; instead, three-dimensional structural overlaps with other enzymes of known function suggested that SPy1599 contains a phosphate-binding pocket in the active site and has possible 6-phospho-ß-glycosidase activity. Subsequent kinetic analysis indeed showed that SPy1599 has 6-phospho-ß-glucosidase (EC 3.2.1.86) activity. These data suggest that SPy1599 is involved in the intracellular degradation of 6-phosphoglycosides, which are likely to originate from import through one of the organism's many phosphoenolpyruvate phosphotransfer systems (PEP-PTSs).

The reaction coordinate of a bacterial GH47 α-mannosidase: a combined quantum mechanical and structural approach.

Mannosides in the southern hemisphere: Conformational analysis of enzymatic mannoside hydrolysis informs strategies for enzyme inhibition and inspires solutions to mannoside synthesis. Atomic resolution structures along the reaction coordinate of an inverting α-mannosidase show how the enzyme distorts the substrate and transition state. QM/MM calculations reveal how the free energy landscape of isolated α-D-mannose is molded on enzyme to only allow one conformationally accessible reaction coordinate.

Life cycle analysis of the model organism Rhodopirellula baltica SH 1(T) by transcriptome studies.

The marine organism Rhodopirellula baltica is a representative of the globally distributed phylum Planctomycetes whose members exhibit an intriguing lifestyle and cell morphology. The analysis of R. baltica's genome has revealed many biotechnologically promising features including a set of unique sulfatases and C1-metabolism genes. Salt resistance and the potential for adhesion in the adult phase of the cell cycle were observed during cultivation. To promote the understanding of this model organism and to specify the functions of potentially useful genes, gene expression throughout a growth curve was monitored using a whole genome microarray approach. Transcriptional profiling suggests that a large number of hypothetical proteins are active within the cell cycle and in the formation of the different cell morphologies. Numerous genes with potential biotechnological applications were found to be differentially regulated, revealing further characteristics of their functions and regulation mechanisms. More specifically, the experiments shed light on the expression patterns of genes belonging to the organism's general stress response, those involved in the reorganization of its genome and those effecting morphological changes. These transcriptomic results contribute to a better understanding of thus far unknown molecular elements of cell biology. Further, they pave the way for the biotechnological exploitation of R. baltica's distinctive metabolic features as a step towards sourcing the phylum Planctomycetes at large.

Expression, purification, crystallization and preliminary X-ray analysis of the polysaccharide lyase RB5312 from the marine planctomycete Rhodopirellula baltica.

Polysaccharide lyases belonging to family PL1 act on pectins. These anionic polymers are usually produced by terrestrial plants and therefore pectinolytic enzymes are not frequently observed in marine microorganisms. The protein RB5312 from the marine bacterium Rhodopirellula baltica is distantly related to family PL1 pectate lyases, but its exact function is unclear. In this study, the expression and purification of a recombinant form of RB5312 are described. This protein was crystallized using the hanging-drop vapour-diffusion method. The crystals belongs to space group P2(1)2(1)2(1), with unit-cell parameters a = 39.05, b = 144.05, c = 153.97 A, alpha = beta = gamma = 90 degrees. A complete data set was collected to 1.8 A resolution from a native crystal.