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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 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 How to find soluble proteins : a comprehensive analysis of alpha/beta hydrolases for recombinant expression in E. coli(2005) Koschorreck, Markus; Fischer, Markus; Barth, Sandra; Pleiss, JürgenBackground: In screening of libraries derived by expression cloning, expression of active proteinsin E. coli can be limited by formation of inclusion bodies. In these cases it would be desirable to enrich gene libraries for coding sequences with soluble gene products in E. coli and thus to improve the efficiency of screening. Previously Wilkinson and Harrison showed that solubility can be predicted from amino acid composition (Biotechnology 1991, 9(5):443-448). We have applied this analysis to members of the alpha/beta hydrolase fold family to predict their solubility in E. coli. alpha/beta hydrolases are a highly diverse family with more than 1800 proteins which have been grouped into homologous families and superfamilies. Results: The predicted solubility in E. coli depends on hydrolase size, phylogenetic origin of the host organism, the homologous family and the superfamily, to which the hydrolase belongs. In general small hydrolases are predicted to be more soluble than large hydrolases, and eukaryotic hydrolases are predicted to be less soluble in E. coli than prokaryotic ones. However, combining phylogenetic origin and size leads to more complex conclusions. Hydrolases from prokaryotic, fungal and metazoan origin are predicted to be most soluble if they are of small, medium and large size, respectively. We observed large variations of predicted solubility between hydrolases from different homologous families and from different taxa. Conclusion: A comprehensive analysis of all alpha/beta hydrolase sequences allows more efficient screenings for new soluble alpha/beta hydrolases by the use of libraries which contain more soluble gene products. Screening of hydrolases from families whose members are hard to express as soluble proteins in E. coli should first be done in coding sequences of organisms from phylogenetic groups with the highest average of predicted solubility for proteins of this family. The tools developed here can be used to identify attractive target genes for expression using protein sequences published in databases. This analysis also directs the design of degenerate, family- specific primers to amplify new members from homologous families or superfamilies with a high probability of soluble alpha/beta hydrolases.Item Open Access Screening, cloning and biochemical characterisation of novel esterases from bacillus sp. associated with the marine sponge aplysina aerophoba(2005) Karpushova, Anna Alexandrovna; Brümmer, Franz; Lange, Stefan; Schmid, Rolf D.Two novel esterases (EstB1 and EstB2) were isolated from a genomic library of Bacillus sp. associated with the marine sponge Aplysina aerophoba. EstB1 shows low identity (26-44 %)with the published hydrolases of the genus Bacillus, whereas EstB2 shows high identity (73-74 %) with the carboxylesterases from B. cereus and B. anthracis. Both esterases were efficiently expressed in Escherichia coli under the control of T7 promoter using the vector pET-22b(+). Recombinant EstB1 was purified in a single step to electrophoretic homogeneity by IMAC. A method for the refolding of inclusion bodies formed by the recombinant EstB2 was established to obtain active enzyme. Substrate specificity of the two enzymes towards pnitrophenyl and methyl esters and the respective kinetic parameters Km and Vmax were determined. The temperature optima of EstB1 and EstB2 were determined to be in the range of 30-50°C and 20-35°C, respectively. The pH optima were found to be in the range of 6.5-7.5 and 6.5-8.0, respectively. Both enzymes showed the highest stability in up to 50 % (v/v) DMSO followed by methanol, ethanol and 2-propanol. The influence of high NaCl and KCl concentrations was tested. The inhibition effect of 10-50 mM Zn2+ and 50 mM Mg2+ and Ca2+ ions was observed for both esterases. 1-5 mM PMSF deactivated the enzymes, whereas β-mercaptoethanol, DTT and EDTA had no effect on the enzymes activity.Item Open Access Recombinant production of human microsomal cytochrome P450 2D6 in the methylotrophic yeast Pichia pastoris(2005) Dietrich, Matthias; Grundmann, Lisa; Kurr, Katja; Valinotto, Laura; Richter, Tanja; Schmid, Rolf D.; Lange, StefanMicrosomal cytochrome P450 monooxygenases of the groups 1 – 3 are mainly expressed in liver and play a crucial role in phase 1 reactions of the xenobiotics metabolism. The cDNAs encoding human CYP2D6 and human NADPH-P450 oxidoreductase (CPR) were transformed into the methylotrophic yeast Pichia pastoris and expressed under control of the methanol inducible AOX1 promotor. The determined molecular weights of the recombinant CYP2D6 and CPR closely matched the calculated values of 55.8 and 76.6 kDa. CPR activity was detected by conversion of cytochrome c using isolated microsomes. Nearly all the recombinant CYP is composed of the active holo-enzyme as confirmed by reduced CO-difference spectra which showed a single peak at 450 nm. Only by co-expression of human CPR and CYP, CYP2D6 activity was obtained. Microsomes containing human CPR and CYP2D6 converted different substrates, such as 3-cyano-7-ethoxycoumarin, parathion, and dextrometorphan. The kinetic parameters of the dextrometorphan conversion closely matched those of CYP2D6 from other recombinant expression systems as well as from human microsomes. The endogenous NADPH-P450 oxidoreductase of Pichia pastoris seems to be incompatible with human CYP2D6, as expression of CYP2D6 without human CPR did not result in any CYP-activity. These recombinant strains provide a novel, easy-to-handle and cheap source for the biochemical characterization of single microsomal cytochromes as well as their allelic variants.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 Biocatalysts for the epoxidation and hydroxylation of fatty acids and fatty alcohols(2005) Maurer, Steffen Christian; Schmid, Rolf D.Whereas most products of the chemical industry are based on petrochemical feedstocks, considerable efforts have been made during the past few decades to use renewable resources as industrial raw materials. Besides polysaccharides and sugars, plant oils and animal fats play an important role in such programmes because of their ready availability (present production is >100 million t/a, which could be increased on demand) of which the lion’s share is used for nutrition (~85 million t/a), whereas ~ 15 – 20 million t/a are used for the synthesis of polymers, surfactants, emollients, lubricants, bio-diesel, emulsifiers, etc.). From a chemical point of view, most natural triglycerides offer just two reactive sites, the ester group and the double bonds of unsaturated fatty acids. In fact, the chemistry of fats and oils is largely focused on the ester group which can be hydrolyzed or catalytically reduced, leading to glycerol and fatty acids or fatty alcohols, respectively. Reactions involving the alkyl chain or double bonds of triglycerides, fatty acids, fatty alcohols or their derivatives represent far less than 10% of today’s oleochemistry, with the production of sulfonated fatty alcohols and their derivatives being a major process of this kind. Oxidation reactions at the alkyl or alkenyl chains would be highly desirable as they would lead to oleochemicals with new properties, but the methods available today lack selectivity and require harsh conditions. Notable exceptions are the epoxidation of unsaturated plant oils and the synthesis and use of a few hydroxy fatty acids.