Biomedical subjects

Pawel Sikorski

Publications and source records attributed to Pawel Sikorski.

10 recordsLinked to original sources

PubMed2006

Costs and benefits of processivity in enzymatic degradation of recalcitrant polysaccharides.

Many enzymes that hydrolyze insoluble crystalline polysaccharides such as cellulose and chitin guide detached single-polymer chains through long and deep active-site clefts, leading to processive (stepwise) degradation of the polysaccharide. We have studied the links between enzyme efficiency and processivity by analyzing the effects of mutating aromatic residues in the substrate-binding groove of a processive chitobiohydrolase, chitinase B from Serratia marcescens. Mutation of two tryptophan residues (Trp-97 and Trp-220) close to the catalytic center (subsites +1 and +2) led to reduced processivity and a reduced ability to degrade crystalline chitin, suggesting that these two properties are linked. Most remarkably, the loss of processivity in the W97A mutant was accompanied by a 29-fold increase in the degradation rate for single-polymer chains as present in the soluble chitin-derivative chitosan. The properties of the W220A mutant showed a similar trend, although mutational effects were less dramatic. Processivity is thought to contribute to the degradation of crystalline polysaccharides because detached single-polymer chains are kept from reassociating with the solid material. The present results show that this processivity comes at a large cost in terms of enzyme speed. Thus, in some cases, it might be better to focus strategies for enzymatic depolymerization of polysaccharide biomass on improving substrate accessibility for nonprocessive enzymes rather than on improving the properties of processive enzymes.

Chitinases↗

PubMed2006

Diffraction to study protein and peptide assemblies.

Proteins and peptides are able to self-assemble in vivo and in vitro. In vitro, this ability can be exploited to make bionanomaterials with many potential uses. Peptides are capable of forming a wide range of structures including fibres, tubules and scaffolds. In vivo, proteins assemble to form cellular fibrous proteins, as well as being involved in protein misfolding in disease. Recent advances using X-ray diffraction have highlighted the internal structure of self-assembled proteins and peptides, showing packing of side chain residues and have enabled a deeper understanding of mechanisms of assembly.

Animals↗

PubMed2006

Serratia marcescens chitinases with tunnel-shaped substrate-binding grooves show endo activity and different degrees of processivity during enzymatic hydrolysis of chitosan.

The modes of action of three family 18 chitinases (ChiA, ChiB, and ChiC) from Serratia marcescens during the degradation of a water-soluble polymeric substrate, chitosan, were investigated using a combination of viscosity measurements, reducing end assays, and characterization of the size-distribution of the oligomeric products. All three enzymes yielded a fast reduction in molecular weight of the chitosan substrate at a very early stage of hydrolysis, which is typical for endo-acting enzymes. For ChiA and ChiB, this is inconsistent with the previously proposed exo-attack mode of action. The main difference between ChiA, ChiB, and ChiC is the degree of processivity. ChiC is an endo enzyme with no apparent processivity. ChiA and ChiB are processive enzymes in which the substrate remains bound to the active cleft after successful hydrolysis and is moved along for the next hydrolysis to occur. ChiA and ChiB perform on average 9.1 and 3.4 cleavages, respectively, for the formation of each enzyme-substrate complex. ChiA and ChiB have deep, tunnel-like substrate-binding grooves. The demonstration of endo activity shows that substrate binding must involve the temporary restructuring of the loops that make up the roofs of the substrate-binding grooves, similar to what has been proposed for cellobiohydrolase Cel6A. The data suggest that the exo-type of activity observed for ChiA and ChiB during the degradation of solid crystalline chitin is due to the better accessibility of chain ends, rather than intrinsic enzyme properties.

Bacterial Proteins↗

PubMed2006

Endo/exo mechanism and processivity of family 18 chitinases produced by Serratia marcescens.

We present a comparative study of ChiA, ChiB, and ChiC, the three family 18 chitinases produced by Serratia marcescens. All three enzymes eventually converted chitin to N-acetylglucosamine dimers (GlcNAc2) and a minor fraction of monomers. ChiC differed from ChiA and ChiB in that it initially produced longer oligosaccharides from chitin and had lower activity towards an oligomeric substrate, GlcNAc6. ChiA and ChiB could convert GlcNAc6 directly to three dimers, whereas ChiC produced equal amounts of tetramers and dimers, suggesting that the former two enzymes can act processively. Further insight was obtained by studying degradation of the soluble, partly deacetylated chitin-derivative chitosan. Because there exist nonproductive binding modes for this substrate, it was possible to discriminate between independent binding events and processive binding events. In reactions with ChiA and ChiB the polymer disappeared very slowly, while the initially produced oligomers almost exclusively had even-numbered chain lengths in the 2-12 range. This demonstrates a processive mode of action in which the substrate chain moves by two sugar units at a time, regardless of whether complexes formed along the way are productive. In contrast, reactions with ChiC showed rapid disappearance of the polymer and production of a continuum of odd- and even-numbered oligomers. These results are discussed in the light of recent literature data on directionality and synergistic effects of ChiA, ChiB and ChiC, leading to the conclusion that ChiA and ChiB are processive chitinases that degrade chitin chains in opposite directions, while ChiC is a nonprocessive endochitinase.

Chitin↗

PubMed2005

Application of high-performance anion-exchange chromatography with pulsed amperometric detection and statistical analysis to study oligosaccharide distributions--a complementary method to investigate the structure and some properties of alginates.

Alginates comprised of essentially alternating units of mannuronic (M) acid-guluronic (G) acid (MG-alginate), and G-blocks isolated from a seaweed where subjected to partial acid hydrolysis at pH 3.5 The chain-length distribution of oligosaccharides in the hydrolysate were investigated by statistical analysis after their separation with high-performance anion-exchange chromatography and pulsed amperometric detection (HPAEC-PAD). Simulated depolymerisation of the MG-alginate provided an estimate of the ratio between two acid hydrolysis rate constants (p=8.3+/-1) and the average distribution of the MM linkages in the original sample of polysaccharide chains. In conclusion, we found HPAEC-PAD together with statistical analysis was a useful method to investigate the fine structure and some properties of binary polysaccharides.

Alginates↗

PubMed2005

Development and application of a model for chitosan hydrolysis by a family 18 chitinase.

Hydrolysis of partially deacetylated chitosans by ChitinaseB (ChiBeta) from Serratia marcescens results in mixtures of oligosaccharides typically between 2 and 20 sugar residues. The amounts of different oligomer fractions depend on the degree of acetylation of the starting chitosans. We have used experimentally determined distributions of hydrolysis products to develop a model for chitosan hydrolysis by ChiB. Important elements of the model include interaction parameters for acetylated/deacetylated units in each of the six subsites in the active cleft and degree of processivity (multiple attack). The hydrolysis reaction is described as a chemical reaction with an activation barrier that depends on the substrate sequence presented to the enzyme subsites. Using a Monte Carlo approach, the interaction parameters were refined by minimizing the difference between observed and predicted amounts of hydrolysis products obtained upon degradation of chitosan with a degree of acetylation of 65%. The final model can accurately predict complex patterns of oligosaccharides produced in the hydrolysis of chitosans with various degrees of acetylation, as well as patterns observed during reactions with chito-oligosaccharides. The behavior of a ChiB mutant with a mutation in subsite +2 (Gly188Asp), which reduces the affinity for an acetylated sugar, could be predicted correctly by introducing one single change in the model parameters (the interaction energy for an acetylated unit in the +2 subsite). The proposed model may be used to explore degradation products for different enzyme-substrates combinations and to optimize conditions for preparation of specific oligosaccharides. In addition, the model provides insight into subsite interaction parameters and the degree of processivity, which complements previous experimental studies on the mode of action of ChiB.

Aspartic Acid↗

PubMed2005

Molecular basis for amyloid fibril formation and stability.

The molecular structure of the amyloid fibril has remained elusive because of the difficulty of growing well diffracting crystals. By using a sequence-designed polypeptide, we have produced crystals of an amyloid fiber. These crystals diffract to high resolution (1 A) by electron and x-ray diffraction, enabling us to determine a detailed structure for amyloid. The structure reveals that the polypeptides form fibrous crystals composed of antiparallel beta-sheets in a cross-beta arrangement, characteristic of all amyloid fibers, and allows us to determine the side-chain packing within an amyloid fiber. The antiparallel beta-sheets are zipped together by means of pi-bonding between adjacent phenylalanine rings and salt-bridges between charge pairs (glutamic acid-lysine), thus controlling and stabilizing the structure. These interactions are likely to be important in the formation and stability of other amyloid fibrils.

Amyloid↗

PubMed2003

Characterisation of bacterial polysaccharides: steps towards single-molecular studies.

Techniques used in studies of polysaccharides, including chemical composition, linkage pattern, and higher order structures are in constant development. They provide information necessary for understanding of the polysaccharide properties and functions. Here, recent advancements in studies of the polysaccharides at the single-molecule level are highlighted. Over the last few years, single-molecule techniques such as force spectroscopy have improved in sensitivity and can today be used to detect forces in the pN range. In addition, these techniques can be used to investigate properties of single molecules close to physiological conditions. The challenges in the interpretation of the observations are aided by control experiments using well-characterised polysaccharides and by data provided by complementary methods. This field is expected to have increasing impact on the further advancement of the molecular understanding of the role of polysaccharides in various biological processes such as recognition and cell adhesion.

Biochemistry↗

PubMed2003

Structure and texture of fibrous crystals formed by Alzheimer's abeta(11-25) peptide fragment.

Amyloid fibril deposition is central to the pathology of Alzheimer's disease. X-ray diffraction from amyloid fibrils formed from full-length Abeta(1-40) and from a shorter fragment, Abeta(11-25), have revealed cross-beta diffraction fingerprints. Magnetic alignment of Abeta(11-25) amyloid fibrils gave a distinctive X-ray diffraction texture, allowing interpretation of the diffraction data and a model of the arrangement of the peptides within the amyloid fiber specimen to be constructed. An intriguing feature of the structure of fibrillar Abeta(11-25) is that the beta sheets, of width 5.2 nm, stack by slipping relative to each other by the length of two amino acid units (0.70 nm) to form beta ribbons 4.42 nm in thickness. Abeta(1-40) amyloid fibrils likely consist of once-folded hairpins, consistent with the size of the fibers obtained using electron microscopy and X-ray diffraction.

Alzheimer Disease↗

PubMedDate not supplied

New model for crystalline polyglutamine assemblies and their connection with amyloid fibrils.

Based on the interpretation of X-ray diffraction data reported for crystals of the poly-L-glutamine-rich 19-peptide D(2)Q(15)K(2), Perutz et al. (Proc. Natl. Acad. Sci. USA 2002, 99, 5591-5595) proposed that hollow, water-filled nanotubes are the basic structural motif of amyloid fibers. We are able to offer an alternative interpretation for the same X-ray diffraction data. Our proposed structure consists of beta-sheets, limited in size in the chain direction that stack at an intersheet distance of 0.83 nm to form cross-beta crystallites. The beta-sheets are composed of individual D(2)Q(15)K(2) molecules hydrogen bonding together in the a direction. The relatively linear interchain amide hydrogen bonds in this growth direction occur at two sites: (i) between neighboring backbone amides and (ii) between adjacent (glutamine) side chain amides decorating both surfaces of the beta sheet. The polyQ sub-lattice unit cell is orthorhombic with parameters a =0.950 nm, b = 1.660 nm, and c = 0.695 nm; contains two beta-sheet segments; and has a calculated density of 1.54 g cm(-3). A key ingredient in the proposed structure is the locking of the Q side chains by hydrogen bonding, which allow high-density packing. In addition, there is evidence suggesting that the D(2)Q(15)K(2) molecules adopt a once-folded hairpin conformation.

Amyloid beta-Peptides↗