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Item Open Access A model of the pressure dependence of the enantioselectivity of Candida rugosa lipase towards (±)-menthol(2001) Kahlow, Ulrich; Schmid, Rolf D.; Pleiss, JürgenTransesterification of (±)-menthol using propionic acid anhydride and Candida rugosa lipase was performed in chloroform and water at different pressures (1, 10, 50, and 100 bar) to study the pressure dependence of enantioselectivity E. As a result, E significantly decreased with increasing pressure from E=55 (1 bar) to E=47 (10 bar), E=37 (50 bar), and E=9 (100 bar). In order to rationalize the experimental findings, molecular dynamics simulations of Candida rugosa lipase were carried out. Analyzing the lipase geometry at 1, 10, 50, and 100 bar revealed a cavity in the Candida rugosa lipase. The cavity leads from a position on the surface distinct from the substrate binding site to the core towards the active site and is limited by F415 and the catalytic H449. In the crystal structure of the Candida rugosa lipase, this cavity is filled with 6 water molecules. The number of water molecules in this cavity gradually increased with increasing pressure: 6 molecules in the simulation at 1 bar, 10 molecules at 10 bar, 12 molecules at 50 bar, and 13 molecules at 100 bar. Likewise, the volume of the cavity progressively increased from about 1864 ų in the simulation at 1 bar to 2529 ų at 10 bar, 2526 ų at 50 bar, and 2617 ų at 100 bar. At 100 bar, one water molecule slipped between F415 and H449, displacing the catalytic histidine side chain and thus opening the cavity to form a continuous water channel. The rotation of the side chain leads to a decreased distance between the H449-N and the (+)-menthyl-oxygen (non-preferred enantiomer) in the acyl enzyme intermediate, a factor determining the enantioselectivity of the lipase. While the geometry of the preferred enantiomer is similar in all simulations, the geometry of the non-preferred enantiomer gets gradually more reactive. This observation correlates with the gradually decreasing enantioselectivity E.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 Structural basis of stereoselectivity in Candida rugosa lipase-catalyzed hydrolysis of secondary alcohols(2001) Schulz, Tanja; Schmid, Rolf D.; Pleiss, JürgenLipases are widely applied catalysts for highly enantioselective resolution of chiral secondary alcohols. While stereopreference is determined predominantly by the substrate structure, stereoselectivity (enantioselectivity and diastereoselectivity) depends on atomic details of interactions between substrate and lipase. Experimentally obtained stereoselectivity and activity in the hydrolysis of butanoic acid esters of two secondary alcohols with two neighbouring stereocenters by Candida rugosa lipase have been investigated by computer-aided molecular modeling of tetrahedral substrate intermediates in complex with the lipase. Breakdown of this intermediate is considered to be the rate-limiting step. Sterical interactions of stereo isomers with the side chain of catalytic histidine led to different orientations of the imidazole. The distance d(HNε-Oalc) between HNε of the imidazole side chain of catalytic histidine and the alcohol oxygen of the substrate was identified to correlate with the experimentally determined reactivity order of the four stereo isomers. Modelled distances d(HNε-Oalc) were short (≤ 1.8 Å) for RR stereo isomers, which were also experimentally found to be hydrolyzed most rapidly; distances d(HNε-Oalc) were about 2 Å for SS and SR stereo isomers, which were converted at similar rates but at lower rate than RR stereo isomers; finally, distances d(HNε-Oalc) for SR stereo isomers were greater than 4 Å, in accordance with very slow conversion of SR stereo isomers.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 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 Regioselectivity of CYP2B6 : homology modelling, molecular dynamics simulation, docking(2002) Bathelt, Christine; Schmid, Rolf D.; Pleiss, JürgenHuman cytochrome P450 (CYP) 2B6 activates the anticancer prodrug cyclophosphamide (CPA) by 4-hydroxylation. In contrast, the same enzyme catalyzes N-deethylation of a structural isomer, the prodrug ifos-famide (IFA) thus causing severe adverse drug effects. To model the molecular interactions leading to a switch in regioselectivity, the structure of CYP2B6 was modelled based on the structure of rabbit CYP2C5. We mod-elled the lacking 22 residue loop in CYP2C5 between helix F and G (F-G loop) which is not resolved in the X-ray structure by molecular dynamics (MD) simulations using a simulated annealing protocol. The modelled conformation of the loop was validated by unconstrained MD simulations of the complete enzymes (CYP2C5 and CYP2B6) in water for 70 and 120 ps, respectively. The simulations were stable and led to a backbone r.m.s. deviation of 1.7 Å between the two CYPs. The shape of the substrate binding site of CYP2B6 was further analyzed. It consists of three well-defined hydro-phobic binding pockets adjacent to the catalytic heme. Size, shape and hydrophobicity of these pockets was compared to the shapes of the two structurally isomeric substrates. In their preferred orientation in the binding site both substrates fill all three binding pockets without repulsive interactions. The distance to the heme iron is short enough for 4-hydroxylation and N-deethylation to occur for CPA and IFA, respectively. However, if the substrates are docked in the non-preferred orientation (such that 4-hydroxylation and N-deethylation would occur for IFA and CPA, respectively), one pocket is left empty, and clashes were observed between the substrates.