147 research outputs found
Substrate-Assisted Catalysis Unifies Two Families of Chitinolytic Enzymes
No full textHen egg-white lysozyme has long been the paradigm for enzymatic glycosyl hydrolysis with retention of configuration, with a protonated carboxylic acid and a deprotonated carboxylate participating in general acid-base catalysis. In marked contrast, the retaining chitin degrading enzymes from glycosyl hydrolase families 18 and 20 all have a single glutamic acid as the catalytic acid but lack a nucleophile on the enzyme. Both families have a catalytic (βα)8-barrel domain in common. X-ray structures of three different chitinolytic enzymes complexed with substrates or inhibitors identify a retaining mechanism involving a protein acid and the carbonyl oxygen atom of the substrate’s C2 N-acetyl group as the nucleophile. These studies unambiguously demonstrate the distortion of the sugar ring toward a sofa conformation, long postulated as being close to that of the transition state in glycosyl hydrolysis.
ProteinCCD with construct scoring and ranking
No full textThe Protein Crystallographic Construct Design [1] ProteinCCD (https://xtal.nki.nl/ccd/) software we previously described in deliverable 7.2 aims to increase the efficiency of researchers producing soluble protein in amounts suitable for structural studies, by facilitating the design of several truncation constructs of a protein under investigation. ProteinCCD functions as a meta server that collects information from (web-based) external software that predicts from sequence secondary structure, disorder, coiled coils, transmembrane segments, domains and domain linkers. Viewing the protein sequence annotated with the prediction results allows users to interactively choose possible starts and ends for suitable protein constructs and designing primers needed for PCR amplification. ProteinCCD outputs a comprehensive view of all constructs and all primers needed for bookkeeping and/or ordering of the designed primers.
The functionality of ProteinCCD has been extended under 7.1 to a new computational platform allowing a more interactive and efficient interface to the user, and providing new analysis options. These include parallel processing of server requests, more efficient interface for construct design, more cloning methods, an extended collection of existing vectors, local execution of some algorithms for improving response time, new servers for meta-analysis, easy bookkeeping, and better data security.
Working towards the goal of this deliverable, to provide construct scoring and ranking we implemented several features to reach this goal. Automated alignments of the “work” protein to orthologues present in typical model species, are now provided to the user to facilitate better choices for constructs. All constructs can now be given to the users not only as the “native” protein sequence (as before) but also in the specific context of the cloning vector used for production, including purification tags, and the sequence after enzymatic cleavage of the tags. This is important, as each proteins version has different properties. The molecular weight, isoelectric point, and absorption coefficient for every construct is also computed, enabling the users to understand the properties of the produced proteins.
The final goal to rank the chances of successfully producing the proteins, is realized by assessing the chances to produce soluble proteins for each protein. A score from 0-1 is given by different servers, and provided to the users. The constructs can then be ranked according to these scores
Assembly of Protein Complexes by Coexpression in Prokaryotic and Eukaryotic Hosts: an Overview
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Bacterial chitobiase structure provides insight into catalytic mechanism and the basis of Tay-Sachs disease
No full textChitin, the second most abundant polysaccharide on earth, is degraded by chitinases and chitobiases. The structure of Serratia marcescens chitobiase has been refined at 1.9 A resolution. The mature protein is folded into four domains and its active site is situated at the C-terminal end of the central (beta alpha)8-barrel. Based on the structure of the complex with the substrate disaccharide chitobiose, we propose an acid-base reaction mechanism, in which only one protein carboxylate acts as catalytic acid, while the nucleophile is the polar acetamido group of the sugar in a substrate-assisted reaction. The structural data lead to the hypothesis that the reaction proceeds with retention of anomeric configuration. The structure allows us to model the catalytic domain of the homologous hexosaminidases to give a structural rationale to pathogenic mutations that underlie Tay-Sachs and Sandhoff disease
The Structural Binding Mode of the Four Autotaxin Inhibitor Types that Differentially Affect Catalytic and Non-Catalytic Functions
No full textAutotaxin (ATX) is a secreted lysophospholipase D, catalysing the conversion of lysophosphatidylcholine (LPC) to bioactive lysophosphatidic acid (LPA). LPA acts through two families of G protein-coupled receptors (GPCRs) controlling key cellular responses, and it is implicated in many physiological processes and pathologies. ATX, therefore, has been established as an important drug target in the pharmaceutical industry. Structural and biochemical studies of ATX have shown that it has a bimetallic nucleophilic catalytic site, a substrate-binding (orthosteric) hydrophobic pocket that accommodates the lipid alkyl chain, and an allosteric tunnel that can accommodate various steroids and LPA. In this review, first, we revisit what is known about ATX-mediated catalysis, crucially in light of allosteric regulation. Then, we present the known ATX catalysis-independent functions, including binding to cell surface integrins and proteoglycans. Next, we analyse all crystal structures of ATX bound to inhibitors and present them based on the four inhibitor types that are established based on the binding to the orthosteric and/or the allosteric site. Finally, in light of these data we discuss how mechanistic differences might differentially modulate the activity of the ATX-LPA signalling axis, and clinical applications including cancer
Evolution of immunoglobulin-like modules in chitinases: their structural flexibility and functional implications
No full textBackground: Chitinase A from Serratia marcescens is a glycosyl hydrolase consisting of three distinct domains. The N-terminal domain (ChiN domain, amino acids 24–137) has an immunoglobulin-like fold. This ChiN domain is structurally similar to fibronectin type III domains (FnIII domains), which exist in other chitinases, but does not share any sequence similarity with them.Results: Structure comparisons of the ChiN domain and FnIII domains confirm the similar fold, but fail to establish any sequence similarity. Sequence searches and comparisons between ChiN and FnIII domain sequences show a remarkable difference between the two domains in chitinases from an evolutionary point of view. A low temperature structure of chitinase A shows that the ChiN module is flexible with respect to the catalytic body of the protein.Conclusions: We postulate that the ChiN and FnIII domains evolved independently in chitinases which share otherwise homologous catalytic domains. The flexibility of the ChiN domain, together with biochemical knowledge of the function of similar domains, leads us to propose that immunoglobulin-like folds in chitinases are involved in interactions with the chitin chain during catalysis
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