Publications by authors named "Andreas Vogel"

42 Publications

An engineered toluene dioxygenase for a single step biocatalytical production of (-)-(1S,2R)-cis-1,2-dihydro-1,2-naphthalenediol.

J Biotechnol 2021 Jan 28;326:37-39. Epub 2020 Dec 28.

Institute of Technical Biochemistry, University of Stuttgart, Allmandring 31, 70569, Stuttgart, Germany. Electronic address:

cis-1,2-Dihydro-1,2-naphthalenediol (DHND) is a valuable molecule employed for the pharmaceutical synthesis of bioactive compounds, such as bicyclic conduritol analogues. Enantiopure (+)-(1R,2S)-DHND (>98 % ee) is easily biosynthesized through the dearomatizing dihydroxylation of naphthalene, catalyzed by toluene dioxygenase (TDO) from Pseudomonas putida F1. However, the opposite enantiomer (-)-(1S,2R)-DHND could not be directly accessed, neither by chemical synthesis nor via biocatalytic approaches. Herein, we report a one-step biosynthesis of the opposite enantiomer (-)-(1S,2R)-DHND in a recombinant TDO E. coli BW25113 platform. We based on a semi-rational approach to generate a set of TDO variants, targeting exclusively the hotspot position F366, in order to enable an enantiomeric switch in the generated product. Eight out of nine single point variants were active and showed not only an alteration in enantioselectivity, but also generated an enantiomeric excess of the pursued product. Variant TDO outperformed above the rest of the set, enabling the synthesis of (-)-(1S,2R)-DHND not only with an excellent enantiomeric excess of 90 %, but also with an advantageous product formation. A comparative semi-preparative biosynthesis yielded, 287 mg of (+)-(1R,2S)-DHND (>98 % ee) and 101 mg of (-)-(1S,2R)-DHND (90 % ee), when performed in a total volume of 100 mL with TDO wild-type and TDO resting cells, respectively.
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http://dx.doi.org/10.1016/j.jbiotec.2020.12.007DOI Listing
January 2021

Optimization of Alcohol Dehydrogenase for Industrial Scale Oxidation of Lactols.

Biotechnol J 2020 Nov 6;15(11):e2000171. Epub 2020 Sep 6.

InnoSyn B.V., Urmonderbaan 22, Geleen, NL-6167RD, The Netherlands.

Alcohol dehydrogenases (ADH) are widely used to enantioselectively reduce ketones to chiral alcohols, but their application in industrial scale oxidations is rare. Reasons are the need for an NAD(P) cofactor regeneration system, often low performance in oxidative reactions and the limited substrate scope of ADHs. ADHA from Candida magnoliae DSMZ 70638 is identified to efficiently catalyze the regio-selective hydroxy-lactone oxidations to hydroxy-lactones. Hydroxy-lactones are common intermediates in industrial processes to cholesterol lowering (va)statin drugs. A biocatalytic aliphatic hydroxy-lactone oxidation process is developed using pure oxygen as oxidant reaching volumetric productivities of up to 12 g L h , product concentrations of almost 50 g L and 95% reaction yield. For co-factor recycling a previously engineered, water-forming NAD(P)H-oxidase from Streptococcus mutans is used. The process is scaled up to industrial pilot plant scale and it could be demonstrated that ADH catalyzed oxidations can be developed to efficient and safe processes. However, the ADHA wild-type enzyme is not productive enough in chlorolactol oxidation. Therefore, enzyme engineering and multi-parameter screening is successfully applied to optimize the enzyme for the target reaction. The optimized ADHA variant shows a 17-fold higher oxidative activity, a 26°C increased stability and is applied to develop an efficient chlorolactol oxidation process.
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http://dx.doi.org/10.1002/biot.202000171DOI Listing
November 2020

Regioselective biocatalytic self-sufficient Tishchenko-type reaction via formal intramolecular hydride transfer.

Chem Commun (Camb) 2020 Jun;56(47):6340-6343

Department of Chemistry, University of Graz, Heinrichstrasse 28, 8010 Graz, Austria.

A self-sufficient nicotinamide-dependent intramolecular bio-Tishchenko-type reaction was developed. The reaction is catalyzed by alcohol dehydrogenases and proceeds through formal intramolecular hydride transfer on dialdehydes to deliver lactones. Regioselectivity on [1,1'-biphenyl]-2,2'-dicarbaldehyde substrates could be controlled via the electronic properties of the substituents. Preparative scale synthesis provided access to substituted dibenzo[c,e]oxepin-5(7H)-ones.
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http://dx.doi.org/10.1039/d0cc02509gDOI Listing
June 2020

Approaching boiling point stability of an alcohol dehydrogenase through computationally-guided enzyme engineering.

Elife 2020 03 31;9. Epub 2020 Mar 31.

Molecular Enzymology Group, University of Groningen, Groningen, Netherlands.

Enzyme instability is an important limitation for the investigation and application of enzymes. Therefore, methods to rapidly and effectively improve enzyme stability are highly appealing. In this study we applied a computational method (FRESCO) to guide the engineering of an alcohol dehydrogenase. Of the 177 selected mutations, 25 mutations brought about a significant increase in apparent melting temperature (Δ ≥ +3 °C). By combining mutations, a 10-fold mutant was generated with a of 94 °C (+51 °C relative to wild type), almost reaching water's boiling point, and the highest increase with FRESCO to date. The 10-fold mutant's structure was elucidated, which enabled the identification of an activity-impairing mutation. After reverting this mutation, the enzyme showed no loss in activity compared to wild type, while displaying a of 88 °C (+45 °C relative to wild type). This work demonstrates the value of enzyme stabilization through computational library design.
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http://dx.doi.org/10.7554/eLife.54639DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7164962PMC
March 2020

Exploring the sequence variability of polymerization-involved residues in the production of levan- and inulin-type fructooligosaccharides with a levansucrase.

Sci Rep 2019 05 22;9(1):7720. Epub 2019 May 22.

Institute of Organic Chemistry, University of Würzburg, Am Hubland, 97074, Würzburg, Germany.

The connection between the gut microbiome composition and human health has long been recognized, such that the host-microbiome interplay is at present the subject of the so-called "precision medicine". Non-digestible fructooligosaccharides (FOS) can modulate the microbial composition and therefore their consumption occupies a central place in a strategy seeking to reverse microbiome-linked diseases. We created a small library of Bacillus megaterium levansucrase variants with focus on the synthesis of levan- and inulin-type FOS. Modifications were introduced at positions R370, K373 and F419, which are either part of the oligosaccharide elongation pathway or are located in the vicinity of residues that modulate polymerization. These amino acids were exchanged by residues of different characteristics, some of them being extremely low- or non-represented in enzymes of the levansucrase family (Glycoside Hydrolase 68, GH68). F419 seemed to play a minor role in FOS binding. However, changes at R370 abated the levansucrase capacity to synthesize levan-type oligosaccharides, with some mutations turning the product specificity towards neo-FOS and the inulin-like sugar 1-kestose. Although variants retaining the native R370 produced efficiently levan-type tri-, tetra- and pentasaccharides, their capacity to elongate these FOS was hampered by including the mutation K373H or K373L. Mutant K373H, for instance, generated 37- and 5.6-fold higher yields of 6-kestose and 6-nystose, respectively, than the wild-type enzyme, while maintaining a similar catalytic activity. The effect of mutations on the levansucrase product specificity is discussed.
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http://dx.doi.org/10.1038/s41598-019-44211-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6531494PMC
May 2019

A novel cytochrome P450 mono-oxygenase from Streptomyces platensis resembles activities of human drug metabolizing P450s.

Biotechnol Bioeng 2018 09 11;115(9):2156-2166. Epub 2018 Jul 11.

Heinrich Heine University Düsseldorf, Institute of Biochemistry, Düsseldorf, Germany.

Cytochrome P450 mono-oxygenases (P450) are versatile enzymes which play essential roles in C-source assimilation, secondary metabolism, and in degradations of endo- and exogenous xenobiotics. In humans, several P450 isoforms constitute the largest part of phase I metabolizing enzymes and catalyze oxidation reactions which convert lipophilic xenobiotics, including drugs, to more water soluble species. Recombinant human P450s and microorganisms are applied in the pharmaceutical industry for the synthesis of drug metabolites for pharmacokinetics and toxicity studies. Compared to the membrane-bound eukaryotic P450s, prokaryotic ones exhibit some advantageous features, such as high stability and generally easier heterologous expression. Here, we describe a novel P450 from Streptomyces platensis DSM 40041 classified as CYP107L that efficiently converts several commercial drugs of various size and properties. This P450 was identified by screening of actinobacterial strains for amodiaquine and ritonavir metabolizing activities, followed by genome sequencing and expression of the annotated S. platensis P450s in Escherichia coli. Performance of CYP107L in biotransformations of amodiaquine, ritonavir, amitriptyline, and thioridazine resembles activities of the main human metabolizing P450s, namely CYPs 3A4, 2C8, 2C19, and 2D6. For application in the pharmaceutical industry, an E. coli whole-cell biocatalyst expressing CYP107L was developed and evaluated for preparative amodiaquine metabolite production.
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http://dx.doi.org/10.1002/bit.26781DOI Listing
September 2018

Development of microreactors with surface-immobilized biocatalysts for continuous transamination.

N Biotechnol 2018 Dec 18;47:18-24. Epub 2018 May 18.

Faculty of Chemistry and Chemical Technology, University of Ljubljana, Večna pot 113, SI-1000, Ljubljana, Slovenia. Electronic address:

The industrial importance of optically pure compounds has thrown a spotlight on ω-transaminases that have shown a high potential for the synthesis of bioactive compounds with a chiral amine moiety. The implementation of biocatalysts in industrial processes relies strongly on fast and cost effective process development, including selection of a biocatalyst form and the strategy for its immobilization. Here, microscale reactors with selected surface-immobilized amine-transaminase (ATA) in various forms are described as platforms for high-throughput process development. Wild type ATA (ATA-wt) from a crude cell extract, as well as Escherichia coli cells intracellularly overexpressing the enzyme, were immobilized on the surfaces of meander microchannels of disposable plastics by means of reactor surface silanization and glutaraldehyde bonding. In addition, a silicon/glass microchannel reactor was used for immobilization of an ATA-wt, genetically engineered to contain a silica-binding module (SBM) at the N-terminus (N-SBM-ATA-wt), leading to immobilization on the non-modified inner microchannel surface. Microreactors with surface-immobilized biocatalysts were coupled with a quenching system and at-line HPLC analytics and evaluated based on continuous biotransformation, yielding acetophenone and l-alanine. E. coli cells and N-SBM-ATA-wt were efficiently immobilized and yielded a volumetric productivity of up to 14.42 g L h, while ATA-wt small load resulted in two orders of magnitude lower productivity. The miniaturized reactors further enabled in-operando characterization of biocatalyst stability, crucial for successful transfer to a production scale.
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http://dx.doi.org/10.1016/j.nbt.2018.05.004DOI Listing
December 2018

Quantitative Analysis of Hepatitis C NS5A Viral Protein Dynamics on the ER Surface.

Viruses 2018 01 8;10(1). Epub 2018 Jan 8.

Goethe Center for Scientific Computing (G-CSC), Goethe Universität Frankfurt, Kettenhofweg 139, 60325 Frankfurt am Main, Germany.

Exploring biophysical properties of virus-encoded components and their requirement for virus replication is an exciting new area of interdisciplinary virological research. To date, spatial resolution has only rarely been analyzed in computational/biophysical descriptions of virus replication dynamics. However, it is widely acknowledged that intracellular spatial dependence is a crucial component of virus life cycles. The hepatitis C virus-encoded NS5A protein is an endoplasmatic reticulum (ER)-anchored viral protein and an essential component of the virus replication machinery. Therefore, we simulate NS5A dynamics on realistic reconstructed, curved ER surfaces by means of surface partial differential equations (sPDE) upon unstructured grids. We match the in silico NS5A diffusion constant such that the NS5A sPDE simulation data reproduce experimental NS5A fluorescence recovery after photobleaching (FRAP) time series data. This parameter estimation yields the NS5A diffusion constant. Such parameters are needed for spatial models of HCV dynamics, which we are developing in parallel but remain qualitative at this stage. Thus, our present study likely provides the first quantitative biophysical description of the movement of a viral component. Our spatio-temporal resolved ansatz paves new ways for understanding intricate spatial-defined processes central to specfic aspects of virus life cycles.
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http://dx.doi.org/10.3390/v10010028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5795441PMC
January 2018

Draft Genome Sequences of Three Strains Presenting New Candidate Organisms with High Potentials for Specific P450 Cytochromes.

Genome Announc 2017 Jul 13;5(28). Epub 2017 Jul 13.

Department of Molecular Biotechnology, Fraunhofer Institute for Interfacial Engineering and Biotechnology, Stuttgart, Germany

The three strains DSM 40041 DSM 535, and DSM 40063 were described to selectively oxyfunctionalize several drugs. Here, we present their draft genomes to unravel their gene sets encoding promising cytochrome P450 monooxygenases associated with the generation of drug metabolites.
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http://dx.doi.org/10.1128/genomeA.00532-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5511904PMC
July 2017

Impaired coordination of nucleophile and increased hydrophobicity in the +1 subsite shift levansucrase activity towards transfructosylation.

Glycobiology 2017 Aug;27(8):755-765

Institute of Organic Chemistry, University of Würzburg, Würzburg, Am Hubland 97074, Germany.

Bacterial levansucrases produce β(2,6)-linked levan-type polysaccharides using sucrose or sucrose analogs as donor/acceptor substrates. However, the dominant reaction of Bacillus megaterium levansucrase (Bm-LS) is hydrolysis. Single domain levansucrases from Gram-positive bacteria display a wide substrate-binding pocket with open access to water, challenging engineering for transfructosylation-efficient enzymes. We pursued a shift in reaction specificity by either modifying the water distribution in the active site or the coordination of the catalytic acid/base (E352) and the nucleophile (D95), thus affecting the fructosyl-transfer rate and allowing acceptors other than water to occupy the active site. Two serine (173/422) and two water-binding tyrosine (421/439) residues located in the first shell of the catalytic pocket were modified. Library variants of S173, Y421 and S422, which coordinate the position of D95 and E352, show increased transfructosylation (30-200%) and modified product spectra. Substitutions at position 422 have a higher impact on sucrose affinity, while changes at position 173 and 421 have a strong effect on the overall catalytic rate. As most retaining glycoside hydrolases (GHs) Bm-LS catalyzes hydrolysis and transglycosylation via a double displacement reaction involving two-transition states (TS1 and TS2). Hydrogen bonds of D95 with the side chains of S173 and S422 contribute a total of 2.4 kcal mol-1 to TS1 stabilization, while hydrogen bonds between invariant Y421, E352 and the glucosyl C-2 hydroxyl-group of sucrose contribute 2.15 kcal mol-1 stabilization. Changes at Y439 render predominantly hydrolytic variants synthesizing shorter oligosaccharides.
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http://dx.doi.org/10.1093/glycob/cwx050DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5881714PMC
August 2017

Three in One: Temperature, Solvent and Catalytic Stability by Engineering the Cofactor-Binding Element of Amine Transaminase.

Chembiochem 2017 08 13;18(15):1482-1486. Epub 2017 Jun 13.

Division of Biotechnology, Department of Chemistry, Lund University, Naturvetarvägen 14, 221 00, Lund, Sweden.

Amine transaminase (ATA) catalyse enantioselectively the direct amination of ketones, but insufficient stability during catalysis limits their industrial applicability. Recently, we revealed that ATAs suffer from substrate-induced inactivation mechanism involving dissociation of the enzyme-cofactor intermediate. Here, we report on engineering the cofactor-ring-binding element, which also shapes the active-site entrance. Only two point mutations in this motif improved temperature and catalytic stability in both biphasic media and organic solvent. Thermodynamic analysis revealed a higher melting point for the enzyme-cofactor intermediate. The high cofactor affinity eliminates the need for pyridoxal 5'-phosphate supply, thus making large-scale reactions more cost effective. This is the first report on stabilising a tetrameric ATA by mutating a single structural element. As this structural "hotspot" is a common feature of other transaminases it could serve as a general engineering target.
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http://dx.doi.org/10.1002/cbic.201700236DOI Listing
August 2017

Direct observation of isolated Damon-Eshbach and backward volume spin-wave packets in ferromagnetic microstripes.

Sci Rep 2016 Feb 24;6:22117. Epub 2016 Feb 24.

The Hamburg Centre for Ultrafast Imaging (CUI), Luruper Chaussee 149, 22761 Hamburg, Germany.

The analysis of isolated spin-wave packets is crucial for the understanding of magnetic transport phenomena and is particularly interesting for applications in spintronic and magnonic devices, where isolated spin-wave packets implement an information processing scheme with negligible residual heat loss. We have captured microscale magnetization dynamics of single spin-wave packets in metallic ferromagnets in space and time. Using an optically driven high-current picosecond pulse source in combination with time-resolved scanning Kerr microscopy probed by femtosecond laser pulses, we demonstrate phase-sensitive real-space observation of spin-wave packets in confined permalloy (Ni80Fe20) microstripes. Impulsive excitation permits extraction of the dynamical parameters, i.e. phase- and group velocities, frequencies and wave vectors. In addition to well-established Damon-Eshbach modes our study reveals waves with counterpropagating group- and phase-velocities. Such unusual spin-wave motion is expected for backward volume modes where the phase fronts approach the excitation volume rather than emerging out of it due to the negative slope of the dispersion relation. These modes are difficult to excite and observe directly but feature analogies to negative refractive index materials, thus enabling model studies of wave propagation inside metamaterials.
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http://dx.doi.org/10.1038/srep22117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4764955PMC
February 2016

Stochastic formation of magnetic vortex structures in asymmetric disks triggered by chaotic dynamics.

Nat Commun 2014 Dec 17;5:5620. Epub 2014 Dec 17.

1] Center for X-ray Optics, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA [2] Physics Department, University of California, Santa Cruz, California 94056, USA.

The non-trivial spin configuration in a magnetic vortex is a prototype for fundamental studies of nanoscale spin behaviour with potential applications in magnetic information technologies. Arrays of magnetic vortices interfacing with perpendicular thin films have recently been proposed as enabler for skyrmionic structures at room temperature, which has opened exciting perspectives on practical applications of skyrmions. An important milestone for achieving not only such skyrmion materials but also general applications of magnetic vortices is a reliable control of vortex structures. However, controlling magnetic processes is hampered by stochastic behaviour, which is associated with thermal fluctuations in general. Here we show that the dynamics in the initial stages of vortex formation on an ultrafast timescale plays a dominating role for the stochastic behaviour observed at steady state. Our results show that the intrinsic stochastic nature of vortex creation can be controlled by adjusting the interdisk distance in asymmetric disk arrays.
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http://dx.doi.org/10.1038/ncomms6620DOI Listing
December 2014

Wave modes of collective vortex gyration in dipolar-coupled-dot-array magnonic crystals.

Sci Rep 2013 ;3:2262

National Creative Research Initiative Center for Spin Dynamics and Spin-Wave Devices, Nanospinics Laboratory, and Research Institute of Advanced Materials, Department of Materials Science and Engineering, Seoul National University, Seoul 151-744, Republic of Korea.

Lattice vibration modes are collective excitations in periodic arrays of atoms or molecules. These modes determine novel transport properties in solid crystals. Analogously, in periodical arrangements of magnetic vortex-state disks, collective vortex motions have been predicted. Here, we experimentally observe wave modes of collective vortex gyration in one-dimensional (1D) periodic arrays of magnetic disks using time-resolved scanning transmission x-ray microscopy. The observed modes are interpreted based on micromagnetic simulation and numerical calculation of coupled Thiele equations. Dispersion of the modes is found to be strongly affected by both vortex polarization and chirality ordering, as revealed by the explicit analytical form of 1D infinite arrays. A thorough understanding thereof is fundamental both for lattice vibrations and vortex dynamics, which we demonstrate for 1D magnonic crystals. Such magnetic disk arrays with vortex-state ordering, referred to as magnetic metastructure, offer potential implementation into information processing devices.
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http://dx.doi.org/10.1038/srep02262DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3719073PMC
February 2014

Enantioselective oxidation of aldehydes catalyzed by alcohol dehydrogenase.

Angew Chem Int Ed Engl 2012 Sep 31;51(39):9914-7. Epub 2012 Aug 31.

Department of Biotechnology, Delft University of Technology, Delft, The Netherlands.

Teaching old dogs new tricks: Alcohol dehydrogenases (ADHs) may be established redox biocatalysts but they still are good for a few surprises. ADHs can be used to oxidize aldehydes, and this was demonstrated by the oxidative dynamic kinetic resolution of profens. In the presence of a suitable cofactor regeneration system, this reaction can occur with high selectivity.
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http://dx.doi.org/10.1002/anie.201203219DOI Listing
September 2012

Tunable negligible-loss energy transfer between dipolar-coupled magnetic disks by stimulated vortex gyration.

Sci Rep 2011 10;1:59. Epub 2011 Aug 10.

National Creative Research Center for Spin Dynamics & Spin-Wave Devices, and Nanospinics Laboratory, Department of Materials Science and Engineering, Seoul National University, Seoul 151-744, Republic of Korea.

A wide variety of coupled harmonic oscillators exist in nature. Coupling between different oscillators allows for the possibility of mutual energy transfer between them and the information-signal propagation. Low-energy input signals and their transport with negligible energy loss are the key technological factors in the design of information-signal processing devices. Here, utilizing the concept of coupled oscillators, we experimentally demonstrated a robust new mechanism for energy transfer between spatially separated dipolar-coupled magnetic disks - stimulated vortex gyration. Direct experimental evidence was obtained by a state-of-the-art experimental time-resolved soft X-ray microscopy probe. The rate of energy transfer from one disk to the other was deduced from the two normal modes' frequency splitting caused by dipolar interaction. This mechanism provides the advantages of tunable energy transfer rates, low-power input signals and negligible energy loss in the case of negligible intrinsic damping. Coupled vortex-state disks might be implemented in applications for information-signal processing.
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http://dx.doi.org/10.1038/srep00059DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3216546PMC
August 2013

Coupled vortex oscillations in spatially separated permalloy squares.

Phys Rev Lett 2011 Apr 29;106(13):137201. Epub 2011 Mar 29.

Institut für Angewandte Physik und Zentrum für Mikrostrukturforschung, Universität Hamburg, 20355 Hamburg, Germany.

We experimentally study the magnetization dynamics of pairs of micron-sized permalloy squares coupled via their stray fields. The trajectories of the vortex cores in the Landau-domain patterns of the squares are mapped in real space using time-resolved scanning transmission x-ray microscopy. After excitation of one of the vortex cores with a short magnetic-field pulse, the system behaves like coupled harmonic oscillators. The coupling strength depends on the separation between the squares and the configuration of the vortex-core polarizations. Considering the excitation via a rotating in-plane magnetic field, it can be understood that only a weak response of the second vortex core is observed for equal core polarizations.
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http://dx.doi.org/10.1103/PhysRevLett.106.137201DOI Listing
April 2011

Influence of dipolar interaction on vortex dynamics in arrays of ferromagnetic disks.

Phys Rev Lett 2010 Jul 12;105(3):037201. Epub 2010 Jul 12.

Institut für Angewandte Physik und Zentrum für Mikrostrukturforschung, Universität Hamburg,Jungiusstrasse 11, 20355 Hamburg, Germany.

The influence of the magnetostatic interaction on vortex dynamics in arrays of ferromagnetic disks is investigated by means of a broadband ferromagnetic-resonance setup. Transmission spectra reveal a strong dependence of the resonance frequency of vortex-core motion on the ratio between the center-to-center distance and the element size. For a decreasing ratio, a considerable broadening of the absorption peak is observed following an inverse sixth power law. An analogy between the vortex system and rotating dipoles is confirmed by micromagnetic simulations.
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http://dx.doi.org/10.1103/PhysRevLett.105.037201DOI Listing
July 2010

Single-cell high-throughput screening to identify enantioselective hydrolytic enzymes.

Angew Chem Int Ed Engl 2008 ;47(27):5085-8

Institute for Biochemistry and Organic Chemistry, Technische Universität Darmstadt, Petersenstrasse 22, 64287 Darmstadt, Germany.

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http://dx.doi.org/10.1002/anie.200705236DOI Listing
August 2008

Loss of unc45a precipitates arteriovenous shunting in the aortic arches.

Dev Biol 2008 Jun 26;318(2):258-67. Epub 2008 Mar 26.

Tumor Biology Training Program, Georgetown University Medical Center, Washington, DC 20057, USA.

Aortic arch malformations are common congenital disorders that are frequently of unknown etiology. To gain insight into the factors that guide branchial aortic arch development, we examined the process by which these vessels assemble in wild type zebrafish embryos and in kurzschluss(tr12) (kus(tr12)) mutants. In wild type embryos, each branchial aortic arch first appears as an island of angioblasts in the lateral pharyngeal mesoderm, then elaborates by angiogenesis to connect to the lateral dorsal aorta and ventral aorta. In kus(tr12) mutants, angioblast formation and initial sprouting are normal, but aortic arches 5 and 6 fail to form a lumenized connection to the lateral dorsal aorta. Blood enters these blind-ending vessels from the ventral aorta, distending the arteries and precipitating fusion with an adjacent vein. This arteriovenous malformation (AVM), which shunts nearly all blood directly back to the heart, is not exclusively genetically programmed, as its formation correlates with blood flow and aortic arch enlargement. By positional cloning, we have identified a nonsense mutation in unc45a in kus(tr12) mutants. Our results are the first to ascribe a role for Unc45a, a putative myosin chaperone, in vertebrate development, and identify a novel mechanism by which an AVM can form.
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http://dx.doi.org/10.1016/j.ydbio.2008.03.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2483962PMC
June 2008

Metal requirements and phosphodiesterase activity of tRNase Z enzymes.

Biochemistry 2007 Dec 4;46(51):14742-50. Epub 2007 Dec 4.

Molekulare Botanik, Universität Ulm, Albert-Einstein-Allee 11, 89069 Ulm, Germany.

The endonuclease tRNase Z from A. thaliana (AthTRZ1) was originally isolated for its tRNA 3' processing activity. Here we show that AthTRZ1 also hydrolyzes the phosphodiester bond in bis(p-nitrophenyl) phosphate (bpNPP) with a kcat of 7.4 s-1 and a KM of 8.5 mM. We analyzed 22 variants of AthTRZ1 with respect to their ability to hydrolyze bpNPP. This mutational mapping identified fourteen variants that lost the ability to hydrolyze bpNPP and seven variants with reduced activity. Surprisingly, a single amino acid change (R252G) resulted in a ten times higher activity compared to the wild type enzyme. tRNase Z enzymes exist in long and short forms. We show here that in contrast to the short tRNase Z enzyme AthTRZ1, the long tRNase Z enzymes do not have bpNPP hydrolysis activity pointing to fundamental differences in substrate cleavage between the two enzyme forms. Furthermore, we determined the metal content of AthTRZ1 and analyzed the metal requirement for bpNPP hydrolysis. AthTRZ1 shows a high affinity for Zn2+ ions; even upon incubation with metal chelators, 0.76 Zn2+ ions are retained per dimer. In contrast to bpNPP hydrolysis, pre-tRNA processing requires additional metal ions, Mn2+ or Mg2+, as Zn2+ ions alone are insufficient.
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http://dx.doi.org/10.1021/bi7010459DOI Listing
December 2007

Directed evolution and axial chirality: optimization of the enantioselectivity of Pseudomonas aeruginosa lipase towards the kinetic resolution of a racemic allene.

Chem Commun (Camb) 2007 May(19):1913-5

Max-Planck-Institut für Kohlenforschung, Kaiser- Wilhelm-Platz 1, D-45470, MülheimlRuhr, Germany.

Directed evolution of Pseudomonas aeruginosa lipase by the use of combinatorial active site saturation test (CAST) criteria provided a highly enantioselective mutant (Leu162Phe) for kinetic resolution of an axially chiral allene, p-nitrophenyl 4-cyclohexyl-2-methylbuta-2,3-dienoate (E=111); the high enantioselectivity of the Leu162Phe mutant was rationalized by pi-pi stacking.
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http://dx.doi.org/10.1039/b700849jDOI Listing
May 2007

Learning from directed evolution: Further lessons from theoretical investigations into cooperative mutations in lipase enantioselectivity.

Chembiochem 2007 Jan;8(1):106-12

Max-Planck-Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 1, 45470 Mülheim/Ruhr, Germany.

An earlier experimental study, which involved the directed evolution of enantioselective lipase variants from Pseudomonas aeruginosa as catalysts in the hydrolytic kinetic resolution of 2-methyl-decanoic acid p-nitrophenyl ester, provided a mutant with six mutations. Consequently, the selectivity factor was found to increase from E = 1.1 for the wild-type to E = 51 for the best mutant. Only one of the amino acid exchanges in this mutant was found to occur next to the binding pocket, the other mutations being remote. Our previous theoretical analysis with molecular-dynamics simulations helped to unveil the source of enhanced enantioselectivity: a relay mechanism that involves two of the six mutations was shown to induce strong cooperativity. In this investigation, single, double, and triple mutants were constructed and tested as enantioselective catalysts. This study supports our original postulate regarding the relay mechanism, offers further mechanistic insight into the role of individual mutations, and provides mutants that display even higher enantioselectivity (E of up to 64).
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http://dx.doi.org/10.1002/cbic.200600359DOI Listing
January 2007

Iterative saturation mutagenesis on the basis of B factors as a strategy for increasing protein thermostability.

Angew Chem Int Ed Engl 2006 Nov;45(46):7745-51

Max-Planck-Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 1, 45470 Mülheim/Ruhr, Germany.

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http://dx.doi.org/10.1002/anie.200602795DOI Listing
November 2006

Expanding the substrate scope of enzymes: combining mutations obtained by CASTing.

Chemistry 2006 Aug;12(23):6031-8

Max-Planck-Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 1, 45470 Mülheim/Ruhr, Germany.

In a previous paper, the combinatorial active-site saturation test (CAST) was introduced as an effective strategy for the directed evolution of enzymes toward broader substrate acceptance. CASTing comprises the systematic design and screening of focused libraries around the complete binding pocket, but it is only the first step of an evolutionary process because only the initial libraries of mutants are considered. In the present study, a simple method is presented for further optimization of initial hits by combining the mutational changes obtained from two different libraries. Combined lipase mutants were screened for hydrolytic activity against six notoriously difficult substrates (bulky carboxylic acid esters) and improved mutants showing significantly higher activity were identified. The enantioselectivity of the mutants in the hydrolytic kinetic resolution of two substrates was also studied, with the best mutant-substrate combination resulting in a selectivity factor of E=49. Finally, the catalytic profile of the evolved mutants in the hydrolysis of simple nonbranched carboxylic acid esters, ranging from acetate to palmitate, was studied for theoretical reasons.
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http://dx.doi.org/10.1002/chem.200600459DOI Listing
August 2006

The crystal structure of the zinc phosphodiesterase from Escherichia coli provides insight into function and cooperativity of tRNase Z-family proteins.

J Bacteriol 2006 Feb;188(4):1607-14

EMBL Hamburg Outstation c/o DESY, Notkestrasse 85, D-22603 Hamburg, Germany.

The elaC gene product from Escherichia coli, ZiPD, is a 3' tRNA-processing endonuclease belonging to the tRNase Z family of enzymes that have been identified in a wide variety of organisms. In contrast to the elaC homologue from Bacillus subtilis, E. coli elaC is not essential for viability, and although both enzymes process only precursor tRNA (pre-tRNA) lacking a CCA triplet at the 3' end in vitro, the physiological role of ZiPD remains enigmatic because all pre-tRNA species in E. coli are transcribed with the CCA triplet. We present the first crystal structure of ZiPD determined by multiple anomalous diffraction at a resolution of 2.9 A. This structure shares many features with the tRNase Z enzymes from B. subtilis and Thermotoga maritima, but there are distinct differences in metal binding and overall domain organization. Unlike the previously described homologous structures, ZiPD dimers display crystallographic symmetry and fully loaded metal sites. The ZiPD exosite is similar to that of the B. subtilis enzyme structurally, but its position with respect to the protein core differs substantially, illustrating its ability to act as a clamp in binding tRNA. Furthermore, the ZiPD crystal structure presented here provides insight into the enzyme's cooperativity and assists the ongoing attempt to elucidate the physiological function of this protein.
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http://dx.doi.org/10.1128/JB.188.4.1607-1614.2006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1367222PMC
February 2006

The tRNase Z family of proteins: physiological functions, substrate specificity and structural properties.

Biol Chem 2005 Dec;386(12):1253-64

Max-Planck-Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 1, D-45470 Mülheim, Germany.

tRNase Z is the endoribonuclease that generates the mature 3'-end of tRNA molecules by removal of the 3'-trailer elements of precursor tRNAs. This enzyme has been characterized from representatives of all three domains of life (Bacteria, Archaea and Eukarya), as well as from mitochondria and chloroplasts. tRNase Z enzymes come in two forms: short versions (280-360 amino acids in length), present in all three kingdoms, and long versions (750-930 amino acids), present only in eukaryotes. The recently solved crystal structure of the bacterial tRNase Z provides the structural basis for the understanding of central functional elements. The substrate is recognized by an exosite that protrudes from the main protein body and consists of a metallo-beta-lactamase domain. Cleavage of the precursor tRNA occurs at the binuclear zinc site located in the other subunit of the functional homodimer. The first gene of the tRNase Z family was cloned in 2002. Since then a comprehensive set of data has been acquired concerning this new enzyme, including detailed functional studies on purified recombinant enzymes, mutagenesis studies and finally the determination of the crystal structure of three bacterial enzymes. This review summarizes the current knowledge about these exciting enzymes.
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http://dx.doi.org/10.1515/BC.2005.142DOI Listing
December 2005

Analysis of the functional modules of the tRNA 3' endonuclease (tRNase Z).

J Biol Chem 2005 Oct 22;280(42):35440-7. Epub 2005 Aug 22.

Molekulare Botanik, Universität Ulm, Albert-Einstein-Allee 11, 89069 Ulm, Germany.

tRNA 3' processing is one of the essential steps during tRNA maturation. The tRNA 3'-processing endonuclease tRNase Z was only recently isolated, and its functional domains have not been identified so far. We performed an extensive mutational study to identify amino acids and regions involved in dimerization, tRNA binding, and catalytic activity. 29 deletion and point variants of the tRNase Z enzyme were generated. According to the results obtained, variants can be sorted into five different classes. The first class still had wild type activity in all three respects. Members of the second and third class still formed dimers and bound tRNAs but had reduced catalytic activity (class two) or no catalytic activity (class three). The fourth class still formed dimers but did not bind the tRNA and did not process precursors. Since this class still formed dimers, it seems that the amino acids mutated in these variants are important for RNA binding. The fifth class did not have any activity anymore. Several conserved amino acids could be mutated without or with little loss of activity.
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http://dx.doi.org/10.1074/jbc.M506418200DOI Listing
October 2005

Structure-function relationships of purple acid phosphatase from red kidney beans based on heterologously expressed mutants.

Arch Biochem Biophys 2005 Aug;440(1):38-45

Institut für Biochemie, Westfälische Wilhelms-Universität Münster, Wilhelm-Klemm-Str. 2, 48149 Münster, Germany.

Purple acid phosphatases are binuclear metalloenzymes, which catalyze the conversion of orthophosphoric monoesters to alcohol and orthophosphate. The enzyme from red kidney beans is characterized with a Fe(III)-Zn(II) active center. So far, the reaction mechanisms postulated for PAPs assume the essentiality of two amino acids, residing near the bimetallic active site. Based on the amino acid sequence of kidney bean PAP (kbPAP), residues H296 and H202 are believed to be essential for catalytic function of the enzyme. In the present study, the role of residue H202 has been elucidated. Mutants H202A and H202R were prepared by site-directed mutagenesis and expressed in baculovirus-infected insect cells. Based on kinetic studies, residue H202 is assumed to play a role in stabilizing the transition state, particularly in charge compensation, steric positioning of the substrate, and facilitating the release of the product by protonating the substrate leaving groups. The study confirmed the essentiality and elucidates the functional role of H202 in the catalytic mechanism of kbPAP.
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http://dx.doi.org/10.1016/j.abb.2005.05.024DOI Listing
August 2005
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