03 Fakultät Chemie
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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 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 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 Reconstitution of beta-carotene hydroxylase activity of thermostable CYP175A1 monooxygenase(2006) Momoi, Kyoko; Hofmann, Ute; Schmid, Rolf D.; Urlacher, Vlada B.CYP175A1 is a thermostable P450 Monooxygenase from Thermus thermophilus HB27, demonstrating in vivo activity towards -carotene. Activity of CYP175A1 was reconstituted in vitro using artificial electron transport proteins. First results were obtained in the mixture with a crude E. coli cell extract at 37°C. In this system -carotene was hydroxylated to -cryptoxanthin. The result indicated the presence of electron transport enzymes among the E. coli proteins, which are suitable for CYP175A1. However, upon in vitro reconstitution of CYP175A1 activity with purified recombinant flavodoxin and flavodoxin reductase from E. coli, only very low -cryptoxanthin production was observed. Remarkably, with another artificial electron transport system, putidaredoxin and putidaredoxin reductase from Pseudomonas putida, purified CYP175A1 enzyme hydroxylated -carotene at 3- and also 3’-positions, resulting in -cryptoxanthin and zeaxanthin. Under the optimal reaction conditions, the turnover rate of the enzyme reached 0.23 nmol -cryptoxanthin produced per nmol P450 per min.Item Open Access Protein engineering of the cytochrome P450 monooxygenase from bacillus megaterium(2004) Urlacher, Vlada B.; Schmid, Rolf D.The role and importance of cytochrome P450 enzymes (CYP) in drug development, biodegradation processes and biocatalysis has been widely acknowledged. P450 monooxygenases exhibit an extremely wide substrate spectrum which is the basis of their ability to activate or detoxify a large variety of target molecules. P450 monooxygenases have been isolated from bacteria, yeasts, insects, as well as mammalian and plant tissues. Currently, the enzyme family is one of the best known gene subfamilies with over 1000 characterized members (http://drnelson.utmem.edu/CytochromeP450.html). Many studies have been dedicated to structural models of cytochrome P450 in order to improve our understanding of the mechanistic details of the enzymes, their substrate specificity and their pronounced stereo- and regiospecificity. In addition, homology modeling of mammalian P450s and QSAR analyses using chemicals which are metabolized by P450s, have added considerably to our understanding of the metabolic variations and functions of the enzyme. Cytochrome P450 enzymes are of considerable interest to pharmaceutical and chemical industry and have thus become targets for protein engineering approaches. Protein engineering is generally defined as the modification of an enzyme by site-directed or random mutagenesis with the aim of altering its properties. Rational design requires a solid structural basis and profound knowledge of the catalytic mechanism of the enzyme which was provided by determining the structures of CYPs using X-ray crystallography at high-resolution. Ten of the twelve crystallized cytochrome P450s are of prokaryotic origin and water-soluble. From a technical point of view, microbial P450s are easier to handle than P450 enzymes from plants and animals. They are not membrane-associated and exhibit a relatively high stability. Eukaryotic cytochrome P450 enzymes are membrane-associated proteins and are hence more difficult to crystallize. Currently, only the X-ray structures of two membrane-bound mammalian P450s, rabbit CYP2C5 and human CYP2C9 are known. Models of other mammalian P450s were built based on the structure of CYP2C5 and its bacterial analogues. P450cam, the cytochrome P450 monooxygenase from Pseudomonas putida, is the best characterized microbial P450 enzyme. In the last fifteen years, a large number of other soluble prokaryotic P450 enzymes have been identified, isolated, subcloned in Escherichia coli, overexpressed and characterized. Cytochrome P450 BM-3 from Bacillus megaterium is catalytically self-sufficient. It contains a P450-heme domain of 54 kDa and an FAD/FMN-reductase domain of 64kDa on a single polypeptide chain. The enzyme catalyzes the subterminal oxidation of saturated and unsaturated fatty acids with a chain length of 12 to 20 carbons. High-resolution X-ray crystal structures are available for substrate-free, palmitic acid-bound and N-palmitoylglycine-bound wild-type and mutant P450 BM-3 enzymes. The structure resolved by NMR is also available. The well-known structure, the availability of the CYP102A1 gene which encodes the protein, and the possibility of expressing the protein in E. coli have encouraged a number of research groups to undertake site-directed mutagenesis studies in order to identify key amino acids. Insights into the mechanisms of P450 BM-3 have been gained and transferred to eukaryotic P450 enzymes. Since P450 BM-3 is an excellent model for addressing questions on the wide substrate specificity of P450s in general and techniques involving the mutagenesis of P450 BM-3 have led to a variety of biocatalysts with features of industrial interests. This review will summarize the recent research on this particular P450 enzyme.Item Open Access Biotransformations using prokaryotic P450 monooxygenases(2002) Urlacher, Vlada B.; Schmid, Rolf D.Recent studies on microbial cytochrome P450 enzymes cover several new areas. Advances have been made in structure-function analysis. New non-enzymatic/electrochemical systems for the replacement of NAD(P)H have been developed. The properties of some enzymes have been re-engineered by site-directed mutagenesis or by methods of directed evolution. New P450s have been functionally expressed and characterized. A combination of these approaches is believed to facilitate the use of isolated P450 monooxygenases in biocatalysis.Item Open Access Immobilisation of P450 BM-3 and an NADP+ cofactor recycling system : towards a technical application of heme-containing monooxygenases in fine chemical synthesis(2003) Maurer, Steffen Christian; Schulze, Holger; Schmid, Rolf D.; Urlacher, Vlada B.Cytochrome P450 monooxygenases are potentially a very useful class of hydroxylation catalysts; they are able to introduce oxygen at activated and non-activated carbon-hydrogen bonds and thus lead to regio- and/or stereochemically pure compounds. However, this potential is lowered by their intrinsic low activity and inherent instability. P450-catalysed biotransformations require a constant supply of NAD(P)H, making the process an expensive one. To render these catalysts more suitable for industrial biocatalysis, the immobilisation of P450 BM-3 (CYP 102A1) from Bacillus megaterium in a sol-gel matrix was combined with a cofactor recycling system based on NADPƒy-dependent formate dehydrogenase (EC 1.2.1.2) from Pseudomonas sp. 101 and tested for practical applicability. This approach was used for the conversion of £]-ionone, octane and naphthalene to the respective hydroxy-compounds with DMSO as cosolvent using sol-gel immobilised P450 BM-3 mutants.Item Open Access Recent advances in oxygenase-catalyzed biotransformations(2006) Urlacher, Vlada B.; Schmid, Rolf D.Oxygenases continue to be widely studied for selective biooxidation of organic compounds. Protein engineering has resulted in heme and flavin monooxygenases with widely altered substrate specificities, and attempts have been reported to scale up reactions catalyzed by these enzymes. Cofactor regeneration is still a key issue in these developments. Protein engineering contributed to understanding of structure vs. function in dioxygenases.