03 Fakultät Chemie
Permanent URI for this collectionhttps://elib.uni-stuttgart.de/handle/11682/4
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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.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 Biotransformation of ionones by engineered cytochrome P450 BM-3(2005) Urlacher, Vlada B.; Makhsumkhanov, Akhmadjan; Schmid, Rolf D.Wild type cytochrome P450 monooxygenase from Bacillus megaterium (P450 BM-3) has low activity for the hydroxylation of beta-ionone (>1 min-1). Substitution of phenylalanine by valine at position 87 increased the beta-ionone hydroxylation activity up to 100-fold (115 min-1). For further activity improvement methods of site-directed and random mutagenesis were applied. The R47L Y51F F87V mutant, designed by site-directed mutagenesis and the A74E F87V P386S mutant, obtained after two rounds of error-prone PCR, exhibit an increase in activity up to 300-fold compared to the wild type enzyme. All mutants converted -ionone regioselectively to 4-hydroxy-beta-ionone.Item Open Access Cloning, expression and characterisation of CYP102A2, a self-sufficient P450 monooxygenase from Bacillus subtilis(2004) Budde, Michael; Maurer, Steffen Christian; Schmid, Rolf D.; Urlacher, Vlada B.The gene encoding CYP102A2, a novel P450 monooxygenase from Bacillus subtilis, was cloned and expressed in Escherichia coli. The recombinant enzyme formed was purified by immobilised metal chelat affinity chromatography (IMAC) and characterised. CYP102A2 is a 119 kDa self-sufficient monooxygenase, consisting of an FMN/FAD–containing reductase domain and a heme domain. The deduced amino acid sequence of CYP102A2 exhibits a high level of identity with the amino acid sequences of CYP102A1 from Bacillus megaterium (59%) and CYP102A3 from Bacillus subtilis (60%). In reduced, CO-bound form, the enzyme shows a typical Soret band at 450 nm. It catalyses the oxidation of even- and odd-chain saturated and unsaturated fatty acids. In all reactions investigated, the products were the respective ù-3, ù-2 and ù-1 hydroxylated fatty acids. Activity was highest towards oleic and linoleic acid (KM=17.4 ± 1.4 ìM, kcat= 2244 ± 72 min-1), linoleic acid (KM=12.25 ± 1.8 ìM, kcat= 1950 ± 84 min-1). Comparison of CYP102A2 homology model to CYP102A1 crystal structure revealed significant differences in the substrate access channels, which might explain the differences in catalytic properties of these two enzymes.Item Open Access Design of acetylcholinesterases for biosensor applications(2003) Schulze, Holger; Vorlová, Sandra; Villatte, Francois; Bachmann, Till T.; Schmid, Rolf D.In recent years, the use of acetylcholinesterases (AChEs) in biosensor technology has gained enormous attention, in particular with respect to insecticide detection. The principle of biosensors using AChE as a biological recognition element is based on the inhibition of the enzyme’s natural catalytic activity by the agent that is to be detected. The advanced understanding of the structure-function-relationship of AChEs serves as the basis for developing enzyme variants, which, compared to the wild type, show an increased inhibition efficiency at low insecticide concentrations and thus a higher sensitivity. This review describes different expression systems that have been used for the production of recombinant AChE. In addition, approaches to purify recombinant AChEs to a degree that is suitable for analytical applications will be elucidated as well as the various attempts that have been undertaken to increase the sensitivity of AChE to specified organophosphates and carbamates using side-directed mutagenesis and employing the enzyme in different assay formats.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 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 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 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.
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