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 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 Rapid detection of neurotoxic insecticides in food using disposable acetylcholinesterase-biosensors and simple solvent extraction(2002) Schulze, Holger; Schmid, Rolf D.; Bachmann, Till T.The extensive use of pesticides to protect agricultural crops necessitates reliable tools for the detection of residues in food and water, thus ensuring environmental protection and consumer safety. Neuroinhibitors such as organophosphates and carbamates in particular, represent a potential hazard to human health. These compounds are frequently found in food but conventional methods of analysis are limited as they are either time consuming or not sufficiently sensitive. As a result, a rapid and sensitive biosensor test based on AChE-inhibition was developed. The disposable AChE-biosensor was directly applied in solvent extracts of food samples using isooctane as extraction solvent. A complete assay could be performed in less than 2 hours. Recovery rates of 84 % were obtained in tests with spiked orange juice samples. Tests in food samples with a lower water content resulted in reduced recovery rates (44 % for peach pap baby food). Phosphorothionate insecticides could be detected after direct oxidation in food with N-bromosuccinimide and solvent extraction. The assay displayed a detection limit of 2 μg/kg paraoxon which was sufficient for the monitoring of maximum residue limits in food according to EU regulations.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 Screen-printed bienzymatic sensor based on sol-gel immobilized Nippostrongylus brasiliensis acetylcholinesterase and a cytochrome P450 BM-3 (CYP102-A1) mutant(2006) Waibel, Michael; Schulze, Holger; Huber, Norbert; Bachmann, Till T.Here we describe the development of a bienzymatic biosensor that simplifies the sample pretreatment steps for insecticide detection, and enables the highly sensitive detection of phosphorothionates in food. These compounds evolve their inhibitory activity towards acetylcholinesterases (AChEs) only after oxidation which is performed in vivo by P450 monooxygenases. Consequently, phosphorothionates require a suitable sample pretreatment by selective oxidation to be detectable in AChE based systems. In this study, enzymatic phosphorothionate activation and AChE inhibition were integrated in a single biosensor unit. A triple mutant of cytochrome P450 BM-3 (CYP 102-A1) and Nippostrongylus brasiliensis AChE (NbAChE) were immobilized using a fluoride catalyzed sol-gel process. Different sol-gel types were fabricated and characterized regarding enzyme loading capacity and enzyme activity containment. The enzyme sol-gel itself already proved to be suitable for the highly sensitive detection of paraoxon and parathion in a spectrometric assay. A method for screen-printing of this enzyme sol-gel on thick film electrodes was developed. Finally, amperometric biosensors containing coimmobilized NbAChE and the cytochrome P450 BM-3 mutant were produced and characterized with respect to signal stability, organophosphate detection, and storage stability. The detection limits achieved were 1 μg/L for paraoxon and 10 μg/L for parathion in its oxidized form, which is according to EC regulations the highest tolerable pesticide concentration in infant food.Item Open Access Microbial P450 enzymes in biotechnology(2004) Urlacher, Vlada B.; Lutz-Wahl, Sabine; Schmid, Rolf D.Oxidations are key reactions in chemical syntheses. Biooxidations using fermentation processes have already conquered some niches in industrial oxidation processes, since they allow the introduction of oxygen even into non-activated carbon atoms in a sterically and optically selective manner which is difficult or impossible to achieve by synthetic organic chemistry. Biooxidation using isolated enzymes is limited to oxidases and dehydrogenases. Surprisingly, cytochrome P450 monooxygenases (CYPs) have scarcely been studied for use in biooxidations, although they are one of the largest known superfamilies of enzyme proteins. Their gene sequences have been identified in various organisms such as humans, bacteria, algae, fungi and plants. The reactions catalyzed by P450s are quite diverse and range from biosynthetic pathways (e.g. those of animal hormones and secondary plant metabolites) to the activation or biodegradation of hydrophobic xenobiotic compounds (e. g. those of various drugs in the liver of higher animals). From a practical point of view, the great potential of P450s is limited by their functional complexity, low activity, and limited stability. In addition, P450-catalyzed reactions require a constant supply of NAD(P)H which makes continuous cell-free processes very expensive. Quite recently, several groups have started to investigate cost-efficient ways which could allow the continuous supply of electrons to the heme iron. These include, for example, the use of electron mediators, direct electron supply from electrodes and enzymatic approaches. In addition, methods of protein design and directed evolution have been applied in an attempt to enhance the activity of the enzymes and improve their selectivity. The promising application of bacterial P450s as catalyzing agents in biocatalytic reactions and recent progress made in this field are covered in this review.Item Open Access Cloning, expression, and characterization of a self-sufficient cytochrome P450 monooxygenase from Rhodococcus ruber DSM 44319(2006) Liu, Luo; Schmid, Rolf D.; Urlacher, Vlada B.A new member of class IV of cytochrome P450 monooxygenases was identified in Rhodococcus ruber strain DSM 44319. As the genome of Rhodococcus ruber has not been sequenced, a P450-like gene fragment was amplified using degenerated primers. The flanking regions of the P450-like DNA fragment were identified by directional genome walking using PCR. The primary protein structure suggests a natural self-sufficient fusion protein consisting of a ferredoxin, flavin-containing reductase and P450 monooxygenase. The only flavin found within the enzyme was FMN. The enzyme was successfully expressed in Escherichia coli and purified and characterized. In the presence of NADPH, the P450 monooxygenase showed hydroxylation activity towards polycyclic aromatic hydrocarbons naphthalene, indene, acenaphthene, toluene, fluorene, m-xylene and ethyl benzene. The conversion of naphthalene, acenaphthene and fluorene resulted in respective ring monohydroxylated metabolites. Alkyl aromatics like toluene, m-xylene and ethyl benzene were hydroxylated exclusively at the side chains. The new enzyme’s ability to oxidize such compounds makes it a potential candidate for biodegradation of pollutants and an attractive biocatalyst for synthesis.Item Open Access Catalytic hydroxylation in biphasic systems using CYP102A1 mutants(2005) Maurer, Steffen Christian; Kühnel, Katja; Kaysser, Leonard A.; Eiben, Sabine; Schmid, Rolf D.; Urlacher, Vlada B.Cytochrome P450 monooxygenases are biocatalysts that hydroxylate or epoxidise a wide range of hydrophobic organic substrates. To date their technical application is limited to a small number of whole-cell biooxidations. The use of the isolated enzymes is believed to be impractical due to the low stability of this enzyme class, to the stochiometric need of the expensive cofactor NADPH, and due to the low solubility of most substrates in aqueous media. To overcome these problems we have investigated the application of a bacterial monooxygenase (mutants of CYP102A1) in a biphasic reaction system supported by cofactor recycling with NADP+-dependent formate dehydrogenase from Pseudomonas sp 101. Using this experimental setup, cyclohexane, octane and myristic acid were hydroxylated. To reduce the process costs a novel NADH-dependent double mutant of CYP102A1 was designed. For recycling of NADH during myristic acid hydroxylation in a biphasic system NAD+-dependent FDH was used. Stability of the monooxygenase under the reaction conditions is quite high as revealed by total turnover numbers of up to 12850 in NADPH-dependent cyclohexane hydroxylation and up to 30000 in NADH-dependent myristic acid oxidation.Item Open Access "In gel patch electrophoresis" : a new method for environmental DNA purification(2005) Roh, Changhyun; Villatte, Francois; Kim, Byung-Gee; Schmid, Rolf D.Most of the microorganism species are largely untapped and could represent an interesting reservoir of genes useful for biotechnological applications. Unfortunately, a major difficulty associated with the methods used to isolate environmental DNA is related to the contamination of the extracted material with humic substances. These polyphenolic compounds inhibit the DNA processing reactions and severely impede cloning procedures. In this work, we describe a rapid, simple and efficient method for the purification of genomic DNA from environmental samples: we added a chromatography step directly embedded into an agarose gel electrophoresis. This strategy enabled the DNA extraction from various environmental samples and it appeared that the purity grade was compatible with digestion by restriction enzymes and PCR amplifications.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.