03 Fakultät Chemie
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Item Open Access Impact of remote mutations on metallo-beta-lactamase substrate specificity : implications for the evolution of antibiotic resistance(2005) Ölschläger, Peter; Mayo, Stephen L.; Pleiss, JürgenMetallo-beta-lactamases have raised concerns due to their ability to hydrolyze a broad spectrum of beta-lactam antibiotics. The G262S point mutation distinguishing the metallo-beta-lactamase IMP 1 from IMP 6 has no effect on the hydrolysis of the drugs cephalothin and cefotaxime, but significantly improves catalytic efficiency toward cephaloridine, ceftazidime, benzylpenicillin, ampicillin, and imipenem. This change in specificity occurs even though residue 262 is remote from the active site. We investigated the substrate specificities of five other point mutants resulting from single nucleotide substitutions at positions near residue 262: G262A, G262V, S121G, F218Y and F218I. The results suggest two types of substrates: type I (nitrocefin, cephalothin and cefotaxime), which are converted equally well by IMP-6, IMP-1, and G262A, but even more efficiently by the other mutants, and type II (ceftazidime, benzylpenicillin, ampicillin, and imipenem), which are hydrolyzed much less efficiently by all the mutants, with IMP-1 being the most active. G262V, S121G, F218Y, and F218I improve conversion of type I substrates, whereas G262A and IMP-1 improve conversion of type II substrates, indicating two distinct evolutionary adaptations from IMP-6. Substrate structure may explain the catalytic efficiencies observed. Type I substrates have R2 electron donors, which may stabilize the substrate intermediate in the binding pocket and lead to enhanced activity. In contrast, the absence of these stabilizing interactions with type II substrates may result in poor conversion and increased sensitivity to mutations. This observation may assist future drug design. As the G262A and F218Y mutants confer effective resistance to Escherichia coli BL21(DE3) cells (high minimal inhibitory concentrations), they are likely to evolve naturally.Item Open Access The Lipase Engineering Database – a navigation and analysis tool for protein families(2003) Fischer, Markus; Pleiss, JürgenThe Lipase Engineering Database (http://www.led.uni-stuttgart.de) integrates information on sequence, structure, and function of lipases, esterases, and related proteins. Sequence data on 806 protein entries are assigned to 38 homologous families, which are grouped into 16 superfamilies with no global sequence similarity between each other. For each family, multisequence alignments are provided with functionally relevant residues annotated. Pre-calculated phylogenetic trees allow navigation inside superfamilies. Experimental structures of 45 proteins are superposed and consistently annotated. The Lipase Engineering Database has been applied to systematically analyze sequence-structure-function relationships of this vast and diverse enzyme class. It is a useful tool to identify functionally relevant residues apart from the active site residues, and to design mutants with desired substrate specificity.Item Open Access Insight into the mechanism of the IMP-1 metallo-beta-lactamase by molecular dynamics simulations(2003) Ölschläger, Peter; Schmid, Rolf D.; Pleiss, JürgenTwo models, a purely nonbonded model and a cationic dummy atom approach, were examined for the modeling of the binuclear zinc-containing IMP-1 metallo-beta-lactamase in complex with a mercaptocarboxylate inhibitor. The cationic dummy atom approach had substantial advantages as it maintained the initial, experimentally determined geometry of the metal-containing active site during molecular dynamics simulations in water. The method was extended to the modeling of the free enzyme and the enzyme in complex with a cephalosporin substrate docked in an intermediate structure. For all three systems, the modeled complexes and the tetrahedral coordination of the zinc ions were stable. The average zinc-zinc distance increased by about 1 Å in the substrate complex compared to the inhibitor complex and the free enzyme in which a hydroxide ion acts as a bridging ligand. Thus, the zinc ions are predicted to undergo a back and forth movement upon the cycle of hydrolysis. In contrast to previous assumptions, no interaction of the Asn167 side chain with the bound cephalosporin substrate was observed. Our observations are in agreement with quantum-mechanical calculations and experimental data and indicate that the cationic dummy atom approach is useful to model zinc-containing metallo-beta-lactamases as free proteins, in complex with inhibitors and in complex with substrates.Item Open Access How to find soluble proteins : a comprehensive analysis of alpha/beta hydrolases for recombinant expression in E. coli(2005) Koschorreck, Markus; Fischer, Markus; Barth, Sandra; Pleiss, JürgenBackground: In screening of libraries derived by expression cloning, expression of active proteinsin E. coli can be limited by formation of inclusion bodies. In these cases it would be desirable to enrich gene libraries for coding sequences with soluble gene products in E. coli and thus to improve the efficiency of screening. Previously Wilkinson and Harrison showed that solubility can be predicted from amino acid composition (Biotechnology 1991, 9(5):443-448). We have applied this analysis to members of the alpha/beta hydrolase fold family to predict their solubility in E. coli. alpha/beta hydrolases are a highly diverse family with more than 1800 proteins which have been grouped into homologous families and superfamilies. Results: The predicted solubility in E. coli depends on hydrolase size, phylogenetic origin of the host organism, the homologous family and the superfamily, to which the hydrolase belongs. In general small hydrolases are predicted to be more soluble than large hydrolases, and eukaryotic hydrolases are predicted to be less soluble in E. coli than prokaryotic ones. However, combining phylogenetic origin and size leads to more complex conclusions. Hydrolases from prokaryotic, fungal and metazoan origin are predicted to be most soluble if they are of small, medium and large size, respectively. We observed large variations of predicted solubility between hydrolases from different homologous families and from different taxa. Conclusion: A comprehensive analysis of all alpha/beta hydrolase sequences allows more efficient screenings for new soluble alpha/beta hydrolases by the use of libraries which contain more soluble gene products. Screening of hydrolases from families whose members are hard to express as soluble proteins in E. coli should first be done in coding sequences of organisms from phylogenetic groups with the highest average of predicted solubility for proteins of this family. The tools developed here can be used to identify attractive target genes for expression using protein sequences published in databases. This analysis also directs the design of degenerate, family- specific primers to amplify new members from homologous families or superfamilies with a high probability of soluble alpha/beta hydrolases.Item Open Access The database of epoxide hydrolases and haloalkane dehalogenases: one structure, many functions(2004) Barth, Sandra; Fischer, Markus; Schmid, Rolf D.; Pleiss, JürgenThe epoxide hydrolases and haloalkane dehalogenase database (EH/HD) integrates sequence and structure of a highly diverse protein family including mainly the Asp-hydrolases of EHs and HDs but also proteins like the Ser-hydrolases non-heme peroxidases, prolyl iminopetidases or 2-hydroxymuconic semialdehyde hydrolases. These proteins have a highly conserved structure, but display a remarkable diversity in sequence and function. 305 protein entries were assigned to 14 homologous families, forming two superfamilies. Annotated multisequence alignments and phylogenetic trees are provided for each homologous family and superfamily. Experimentally derived structures of 19 proteins are superposed and consistently annotated. Sequence and structure of all 305 proteins were systematically analysed. Thus, deeper insight is gained into the role of a highly conserved sequence motifs and structural elements. The EH/HD database is available at http://www.led.uni-stuttgart.de.Item Open Access Structure and dynamics of Candida rugosa lipase : the role of organic solvent(2004) Tejo, Bimo Ario; Abu Bakar Salleh; Pleiss, JürgenThe effect of organic solvent to structure and dynamics of proteins was investigated by multiple molecular dynamics simulations (1 ns each) of Candida rugosa lipase in water and in carbon tetrachloride. The choice of solvent had only a minor structural effect. For both solvents the open and the closed conformation of the lipase were near to their experimental X-ray structures (Cα rms deviation 1-1.3 Å). However, the solvents had a highly specific effect on the flexibility of solvent-exposed side chains: polar side chains were more flexible in water, but less flexible in organic solvent. In contrast, hydrophobic residues were more flexible in organic solvent, but less flexible in water. As a major effect solvent changed the dynamics of the lid, a mobile element involved in activation of the lipase, which fluctuated as rigid body about its average position. While in water the deviations were about 1.6 Å, organic solvent reduced flexibility to 0.9 Å. This increase rigidity was caused by two salt bridges (Lys85-Asp284, Lys75-Asp79) and a stable hydrogen bond (Lys75-Asn 292) in organic solvent. Thus organic solvents stabilize the lid but render the side chains in the hydrophobic substrate binding site more mobile.Item Open Access Sequence and structure of epoxide hydrolases : a systematic analysis(2004) Barth, Sandra; Fischer, Markus; Schmid, Rolf D.; Pleiss, JürgenEpoxide hydrolases (EC 3.3.2.3) are ubiquitous enzymes which catalyze the hydrolysis of epoxides to the corresponding vicinal diols. Over 100 epoxide hydrolases (EH) have been identified or predicted, 3 structures are available. Although they catalyze the same chemical reaction, sequence similarity is low. To identify conserved regions, all EHs were aligned. Phylogenetic analysis identified 12 homologous families, which were grouped into 2 major superfamilies: the microsomal EH superfamily, which includes the homologous families of Mammalian, Insect, Fungal, and Bacterial EHs, and the cytosolic EH superfamily, which includes Mammalian, Plant, and Bacterial EHs. Bacterial EHs show a high sequence diversity. Based on structure comparison of 3 known structures from Agrobacterium radiobacter AD1 (cytosolic EH), Aspergillus niger (microsomal EH), and Mus musculus (cytosolic EH), and multisequence alignment and phylogenetic analysis of 95 EHs, the modular architecture of this enzyme family was analyzed. While core and cap domain are highly conserved, the structural differences between the EHs are restricted to only 2 loops: the NC-loop connecting the core and the cap and the cap-loop which is inserted into the cap domain. EHs were assigned to either of 3 clusters based on loop length. Using this classification, core and cap region of all EHs, NC-loops and cap-loops of 78% and 89% of all EHs, respectively, could be modeled. Representative models are available from the Lipase Engineering Database, http://www.led.uni-stuttgart.de.Item Open Access Blocking the tunnel: engineering of Candida rugosa lipase mutants with short chain length specificity(2002) Schmitt, Jutta; Brocca, Stefania; Schmid, Rolf D.; Pleiss, JürgenThe molecular basis of chain length specificity of Candida rugosa lipase 1 was investigated by molecular modelling and site-directed mutagenesis. The synthetic lip1 gene and the lipase mutants were expressed in Pichia pastoris and assayed for their chain length specificity in single substrate assays using triglycerides as well as in a competitive substrate assay using a randomized oil. Mutation of amino acids at different locations inside the tunnel (P246F, L413F, L410W, L410F/S300E, L410F/S365L) resulted in mutants with a different chain length specificity. Mutants P246F and L413F have a strong preference for short chain lengths whereas substrates longer than C10 are hardly hydrolyzed. Increasing the bulkiness of the amino acid at position 410 led to mutants that show a strong discrimination of chain lengths longer than C14. The results obtained can be explained by a simple mechanical model: the activity for a fatty acid sharply decreases as it becomes long enough to reach the mutated site. In contrast, a mutation at the entrance of the tunnel (L304F) has a strong impact on C4 and C6 substrates. This mutant is nevertheless capable to hydrolyze chain lengths longer than C8.Item Open Access The molecular mechanism of enantiorecognition of tertiary alcohols by carboxylesterases(2003) Henke, Erik; Bornscheuer, Uwe Theo; Schmid, Rolf D.; Pleiss, JürgenCarboxylesterases containing the sequence motif GGGX catalyze hydrolysis of esters of chiral tertiary alcohols, albeit at only low to moderate enantioselectivity towards three model substrates (linalyl acetate, methyl-1-pentin-1-yl acetate, 2-phenyl-3-butin-2-yl acetate). In order to understand the molecular mechanism of enantiorecognition and to improve enantioselectivity towards this interesting substrate class, the interaction of both enantiomers with the substrate binding sites of acetylcholinesterases and p-nitrobenzyl esterase from Bacillus subtilis was modeled and correlated to experimental enantioselectivity. For all substrate-enzyme pairs, enantiopreference and ranking by enantioselectivity could be predicted by the model. In p-nitrobenzyl esterase, one of the key residues in determining enantioselectivity was G105: exchange of this residue by alanine led to a six-fold increase of enantioselectivity (E=19) towards 2-phenyl-3-butin-2-yl acetate. However, the effect of this mutation is personalized: towards the substrate linalyl acetate, the same mutant had a reversed enantiopreference. Thus, depending on the substrate structure, the same mutant had either increased enantioselectivity or opposite enantiopreference compared to wild type enzyme.Item Open Access Molecular modelling of family GH16 glycoside hydrolases : potential roles for xyloglucan endotransglucosylases/hydrolases in cell wall modification in the Poaceae(2004) Strohmeier, Marco; Hrmova, Maria; Fischer, Markus; Harvey, Andrew J.; Pleiss, Jürgen; Fincher, Geoffrey B.Family GH16 glycoside hydrolases can be assigned to five sub-groups according to their substrate specificities, including xyloglucan endotransglucosylases/hydrolases (XTHs), (1,3)-β- galactanases, (1,4)-β-galactanases/κ-carrageenases, “non-specific” (1,3/1,3;1,4)-β-D-glucan endohydrolases and (1,3;1,4)-β-D-glucan endohydrolases. A structured family GH16 glycoside hydrolase database has been constructed (http://www.ghdb.uni-stuttgart.de) and provides multiple sequence alignments with functionally annotated amino acid residues and phylogenetic trees. The database has been used for homology modelling of seven family GH16 glycoside hydrolases, based on structural coordinates for (1,3;1,4)-β-D-glucan endohydrolases and a κ-carrageenase. In combination with multiple sequence alignments, the models predict the three-dimensional dispositions of amino acid residues in the substrate-binding and catalytic sites of XTHs and (1,3/1,3;1,4)-β-D-glucan endohydrolases, for which no structural information is available. Furthermore, they reveal similarities with the active sites of family GH11 (1,4)-β-D-xylan endohydrolases. From a biological viewpoint, the classification and molecular modelling establish structural and evolutionary connections between XTHs, (1,3;1,4)-β-D-glucan endohydrolases and xylan endohydrolases, and raise the possibility that XTHs from higher plants could be active not only on cell wall xyloglucans, but also on (1,3;1,4)-β-D-glucans and arabinoxylans, which are major components of walls in grasses. A role for XTHs in (1,3;1,4)-β-D-glucan and arabinoxylan modification would be consistent with the apparent over-representation of XTH sequences in cereal EST databases.