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 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.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.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 DNA isolation from soil samples for cloning in different hosts(2004) Kauffmann, Isabelle Melanie; Schmitt, Jutta; Schmid, Rolf D.Many protocols to extract DNA directly from soil samples have been developed in recent years. We employed two extraction methods which differed in the method of lysis and compared these methods with respect to yield, purity and degree of shearing. The main focus was on the specific isolation of DNA from different microorganisms, especially DNA from actinomycetes, as these cells are very difficult to lyse in contrast to non-actinomycetes. Thus, we used both methods to isolate DNA from Pseudomonas (Entcheva et al. 2001), Arthrobacter and Rhodococcus (Borneman et al. 1996) and from soil spiked with the respective microorganisms. Both methods rendered high DNA yields with a low degree of shearing but differed in the type of cells that were lysed. By one protocol (utilizing enzymatic lysis) only DNA from the Gram-negative Pseudomonas strain could be obtained whereas by the other protocol (utilizing mechanical lysis), all microorganisms that were used could be lysed and DNA from them extracted. Using a combination of both protocols, DNA from those organisms could be obtained selectively. Furthermore, one of the protocols was modified, resulting in higher DNA yield and purity.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.