Juergen Koepke - Academia.edu (original) (raw)

Papers by Juergen Koepke

Biochimica Et Biophysica Acta-proteins and Proteomics, 2005

Strictosidine β-d-glucosidase, a plant enzyme initiating biosynthetic pathways to about 2000 mono... more Strictosidine β-d-glucosidase, a plant enzyme initiating biosynthetic pathways to about 2000 monoterpenoid indole alkaloids with an extremely large number of various carbon skeletons, has been functionally expressed in Escherichia coli and purified to homogeneity in mg scale. Crystals suitable for X-ray analysis were found by robot-mediated screening. Using the hanging-drop technique, optimum conditions were 0.3 M ammonium sulfate, 0.1 M

Structure, 2000

Results: The cytochrome bc 1 complex from the yeast Saccharomyces cerevisiae was crystallized tog... more Results: The cytochrome bc 1 complex from the yeast Saccharomyces cerevisiae was crystallized together with a bound antibody Fv fragment. The structure was determined at 2.3 Å resolution using multiple isomorphous replacement, and refined to a crystallographic R factor of ...

Acta Crystallographica Section D-biological Crystallography, 2001

Acta crystallographica. Section D, Biological crystallography, 2005

Strictosidine synthase (STR1) is a central enzyme that participates in the biosynthesis of almost... more Strictosidine synthase (STR1) is a central enzyme that participates in the biosynthesis of almost all plant monoterpenoid indole alkaloids. After heterologous expression in Escherichia coli, crystals of STR1 and its substrate complex with tryptamine were obtained by the hanging-drop technique at 302-304 K with potassium sodium tartrate tetrahydrate as precipitant. All crystals belong to space group R3. The native STR1 crystals diffract to 2.95 A and have unit-cell parameters a = b = 150.3, c = 122.4 A. The tryptamine complex crystals diffract to 2.38 A, with unit-cell parameters a = b = 147.3, c = 122.3 A.

Plant Cell, 2006

The enzyme strictosidine synthase (STR1) from the Indian medicinal plant Rauvolfia serpentina is ... more The enzyme strictosidine synthase (STR1) from the Indian medicinal plant Rauvolfia serpentina is of primary importance for the biosynthetic pathway of the indole alkaloid ajmaline. Moreover, STR1 initiates all biosynthetic pathways leading to the entire monoterpenoid indole alkaloid family representing an enormous structural variety of ;2000 compounds in higher plants. The crystal structures of STR1 in complex with its natural

THE PLANT CELL ONLINE, 2007

Strictosidine b-D-glucosidase (SG) follows strictosidine synthase (STR1) in the production of the... more Strictosidine b-D-glucosidase (SG) follows strictosidine synthase (STR1) in the production of the reactive intermediate required for the formation of the large family of monoterpenoid indole alkaloids in plants. This family is composed of ;2000

Structure, 2001

Background: Phosphotriesterases (PTE) are enzymes capable of detoxifying organophosphate-based ch... more Background: Phosphotriesterases (PTE) are enzymes capable of detoxifying organophosphate-based chemical warfare agents by hydrolysis. One subclass of these enzymes comprises the family of diisopropylfluorophosphatases (DFPases). The DFPase reported here was originally isolated from squid head ganglion of Loligo vulgaris and can be characterized as squid-type DFPase. It is capable of hydrolyzing the organophosphates diisopropylfluorophosphate, soman, sarin, tabun, and cyclosarin.Results: Crystals

Structure, 1996

The light-harvesting complexes II (LH-2s) are integral membrane proteins that form ring-like stru... more The light-harvesting complexes II (LH-2s) are integral membrane proteins that form ring-like structures, oligomers of alpha beta-heterodimers, in the photosynthetic membranes of purple bacteria. They contain a large number of chromophores organized optimally for light absorption and rapid light energy migration. Recently, the structure of the nonameric LH-2 of Rhodopseudomonas acidophila has been determined; we report here the crystal structure of the octameric LH-2 from Rhodospirillum molischianum. The unveiling of similarities and differences in the architecture of these proteins may provide valuable insight into the efficient energy transfer mechanisms of bacterial photosynthesis. The crystal structure of LH-2 from Rs. molischianum has been determined by molecular replacement at 2.4 A resolution using X-ray diffraction. The crystal structure displays two concentric cylinders of sixteen membrane-spanning helical subunits, containing two rings of bacteriochlorophyll-a (BChl-a) molecules. One ring comprises sixteen B850 BChl-as perpendicular to the membrane plane and the other eight B800 BChl-as that are nearly parallel to the membrane plane; eight membrane-spanning lycopenes (the major carotenoid in this complex) stretch out between the B800 and B850 BChl-as. The B800 BChl-as exhibit a different ligation from that of Rps. acidophila (aspartate is the Mg ligand as opposed to formyl-methionine in Rps. acidophila). The light-harvesting complexes from different bacteria assume various ring sizes. In LH-2 of Rs. molischianum, the Qy transition dipole moments of neighbouring B850 and B800 BChl-as are nearly parallel to each other, that is, they are optimally aligned for Föster exciton transfer. Dexter energy transfer between these chlorophylls is also possible through interactions mediated by lycopenes and B850 BChl-a phytyl tails; the B800 BChl-a and one of the two B850 BChl-as associated with each heterodimeric unit are in van der Waals distance to a lycopene, such that singlet and triplet energy transfer between lycopene and the BChl-as can occur by the Dexter mechanism. The ring structure of the B850 BChl-as is optimal for light energy transfer in that it samples all spatial absorption and emission characteristics and places all oscillator strength into energetically low lying, thermally accessible exciton states.

Protein Science, 1995

We attempted to predict through computer modeling the structure of the light-harvesting complex I... more We attempted to predict through computer modeling the structure of the light-harvesting complex II (LH-Ii) of Rhodospirillum molischianum, before the impending publication of the structure of a homologous protein solved by means of X-ray diffraction. The protein studied is an integral membrane protein of 16 independent polypeptides, 8 a-apoproteins and 8 @-apoproteins, which aggregate and bind to 24 bacteriochlorophyll-a's and 12 lycopenes. Available diffraction data of a crystal of the protein, which could not be phased due to a lack of heavy metal derivatives, served to test the predicted structure, guiding the search. In order to determine the secondary structure, hydropathy analysis was performed to identify the putative transmembrane segments and multiple sequence alignment propensity analyses were used to pinpoint the exact sites of the 20-residue-long transmembrane segment and the 4-residue-long terminal sequence at both ends, which were independently verified and improved by homology modeling. A consensus assignment for the secondary structure was derived from a combination of all the prediction methods used. Three-dimensional structures for the cy-and the @-apoprotein were built by comparative modeling. The resulting tertiary structures are combined, using X-PLOR, into an a@ dimer pair with bacteriochlorophyll-a's attached under constraints provided by site-directed mutagenesis and spectral data. The a@ dimer pairs were then aggregated into a quaternary structure through further molecular dynamics simulations and energy minimization. The structure of LH-I1 so determined is an octamer of a@ heterodimers forming a ring with a diameter of 70 A.

Journal of Structural and Functional Genomics, 2012

Structure determination of membrane proteins and membrane protein complexes is still a very chall... more Structure determination of membrane proteins and membrane protein complexes is still a very challenging field. To facilitate the work on membrane proteins the Core Centre follows a strategy that comprises four labs of protein analytics and crystal handling, covering mass spectrometry, calorimetry, crystallization and X-ray diffraction. This general workflow is presented and a capacity of 20% of the operating time of all systems is provided to the European structural biology community within the ESFRI Instruct program. A description of the crystallization service offered at the Core Centre is given with detailed information on screening strategy, screens used and changes to adapt high throughput for membrane proteins. Our aim is to constantly develop the Core Centre towards the usage of more efficient methods. This strategy might also include the ability to automate all steps from crystallization trials to crystal screening; here we look ahead how this aim might be realized at the Core Centre.

Journal of Molecular Biology, 2007

The structure of the photosynthetic reaction-center from Rhodobacter sphaeroides has been determi... more The structure of the photosynthetic reaction-center from Rhodobacter sphaeroides has been determined at four different pH values (6.5, 8.0, 9.0, 10.0) in the neutral and in charge separated states. At pH 8.0, in the neutral state, we obtain a resolution of 1.87 Å, which is the best ever reported for the bacterial reaction center protein. Our crystallographic data confirm the existence of two different binding positions of the secondary quinone (Q B ). We observe a new orientation of Q B in its distal position, which shows no ring-flip compared to the orientation in the proximal position. Datasets collected for the different pH values show a pH-dependence of the population of the proximal position. The new orientation of Q B in the distal position and the pH-dependence could be confirmed by continuum electrostatics calculations. Our calculations are in agreement with the experimentally observed proton uptake upon charge separation. The high resolution of our crystallographic data allows us to identify new water molecules and external residues being involved in two previously described hydrogen bond proton channels. These extended proton-transfer pathways, ending at either of the two oxogroups of Q B in its proximal position, provide additional evidence that ring-flipping is not required for complete protonation of Q B upon reduction.

Journal of Molecular Biology, 2008

Asparagine 131, located near the cytoplasmic entrance of the D-pathway in subunit I of the Paraco... more Asparagine 131, located near the cytoplasmic entrance of the D-pathway in subunit I of the Paracoccus denitrificans aa 3 cytochrome c oxidase, is a residue crucial for proton pumping. When replaced by an aspartate, the mutant enzyme is completely decoupled: while retaining full cytochrome c oxidation activity, it does not pump protons. The same phenotype is observed for two other substitutions at this position (N131E and N131C), whereas a conservative replacement by glutamine affects both activities of the enzyme. The N131D variant oxidase was crystallized and its structure was solved to 2.32-Å resolution, revealing no significant overall change in the protein structure when compared with the wild type (WT), except for an alternative orientation of the E278 side chain in addition to its WT conformation. Moreover, remarkable differences in the crystallographically resolved chain of water molecules in the D-pathway are found for the variant: four water molecules that are observed in the water chain between N131 and E278 in the WT structure are not visible in the variant, indicating a higher mobility of these water molecules. Electrochemically induced Fourier transform infrared difference spectra of decoupled mutants confirm that the protonation state of E278 is unaltered by these mutations but indicate a distinct perturbation in the hydrogen-bonding environment of this residue. Furthermore, they suggest that the carboxylate side chain of the N131D mutant is deprotonated. These findings are discussed in terms of their mechanistic implications for proton routing through the D-pathway of cytochrome c oxidase.

Biochemistry, 2000

The cytochrome (cyt) subunit of the photosynthetic reaction center from Rhodopseudomonas viridis ... more The cytochrome (cyt) subunit of the photosynthetic reaction center from Rhodopseudomonas viridis contains four heme groups in a linear arrangement in the spatial order heme1, heme2, heme4, and heme3. Heme3 is the direct electron donor to the photooxidized primary electron donor (special pair, P(+)). This heme has the highest redox potential (E(m)) among the hemes in the cyt subunit. The E(m) of heme3 has been specifically lowered by site-directed mutagenesis in which the Arg residue at the position of 264 of the cyt was replaced by Lys. The mutation decreases the E(m) of heme3 from +380 to +270 mV, i.e., below that of heme2 (+320 mV). In addition, a blue shift of the alpha-band was found to accompany the mutation. The assignment of the lowered E(m) and the shifted alpha-band to heme3 was confirmed by spectroscopic measurements on RC crystals. The structure of the mutant RC has been determined by X-ray crystallography. No remarkable differences were found in the structure apart from the mutated residue itself. The velocity of the electron transfer (ET) from the tetraheme cyt to P(+) was measured under several redox conditions by following the rereduction of P(+) at 1283 nm after a laser flash. Heme3 donates an electron to P(+) with t(1/2) = 105 ns, i.e., faster than in the wild-type reaction center (t(1/2) = 190 ns), as expected from the larger driving force. The main feature is that a phase with t(1/2) approximately 2 micros dominates when heme3 is oxidized but heme2 is reduced. We conclude that the ET from heme2 to heme3 has a t(1/2) of approximately 2 micros, i.e., the same as in the WT, despite the fact that the reaction is endergonic by 50 meV instead of exergonic by 60 meV. We propose that the reaction kinetics is limited by the very uphill ET from heme2 to heme4, the DeltaG degrees of which is about the same (+230 meV) in both cases. The interpretation is further supported by measurements of the activation energy (216 meV in the wild-type, 236 meV in the mutant) and by approximate calculations of ET rates. Altogether these results demonstrate that the ET from heme2 to heme3 is stepwise, starting with a first very endergonic step from heme2 to heme4.

Biochemistry, 2005

The active site, the substrate binding site, and the metal binding sites of the diisopropylfluoro... more The active site, the substrate binding site, and the metal binding sites of the diisopropylfluorophosphatase (DFPase) from Loligo vulgaris have been modified by means of site-directed mutagenesis to improve our understanding of the reaction mechanism. Enzymatic characterization of mutants located in the major groove of the substrate binding pocket indicates that large hydrophobic side chains at these positions are favorable for substrate turnover. Moreover, the active site residue His287 proved to be beneficial, but not essential, for DFP hydrolysis. In most cases, hydrophobic side chains at position 287 led to significant catalytic activities although reduced relative to the wild-type enzyme. With respect to the Ca-1 binding site, where catalysis occurs, various mutants indicated that the net charge at this calcium-binding site as well as the relative positions of the charged calcium ligands is crucial for catalytic activity. The importance of the electrostatic potential at the active site was furthermore revealed by various mutations of residues lining the interior of the central water-filled tunnel, which traverses the entire protein structure. In this respect, the structural features of residue His181, which is located at the opposite end of the DFPase tunnel relative to the active site, were characterized extensively. It was concluded that a tunnel-spanning hydrogen bond network, which includes a large number of apparently slow exchanging water molecules, relays any modifications in the electrostatics of the system to the active site, thus affecting the catalytic reactivity of the enzyme.

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2004

Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1990

Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2009

The structure of the two-subunit cytochrome c oxidase from Paracoccus denitrificans has been refi... more The structure of the two-subunit cytochrome c oxidase from Paracoccus denitrificans has been refined using X-ray cryodata to 2.25 Å resolution in order to gain further insights into its mechanism of action. The refined structural model shows a number of new features including many additional solvent and detergent molecules. The electron density bridging the heme a 3 iron and Cu B of the active site is fitted best by a peroxogroup or a chloride ion. Two waters or OH − groups do not fit, one water (or OH − ) does not provide sufficient electron density. The analysis of crystals of cytochrome c oxidase isolated in the presence of bromide instead of chloride appears to exclude chloride as the bridging ligand. In the D-pathway a hydrogen bonded chain of six water molecules connects Asn131 and Glu278, but the access for protons to this water chain is blocked by Asn113, Asn131 and Asn199. The K-pathway contains two firmly bound water molecules, an additional water chain seems to form its entrance. Above the hemes a cluster of 13 water molecules is observed which potentially form multiple exit pathways for pumped protons. The hydrogen bond pattern excludes that the Cu B ligand His326 is present in the imidazolate form.

Acta Crystallographica Section D Biological Crystallography, 2002

Diisopropylfluorophosphatases (DFP-ases) are capable of detoxifying chemical warfare agents like ... more Diisopropylfluorophosphatases (DFP-ases) are capable of detoxifying chemical warfare agents like diisopropylfluorophosphate (DFP) by hydrolysis. The protein reported here was recombinantely expressed in E. coli. The X-ray crystal structure of this enzyme has been refined to a resolution of 0.85 A and a crystallographic R value of 9.4%. Reversible flash-cooling improved both, mosaicity and resolution of the crystals considerably. The overall structure of this protein represents a six-bladed beta-propeller with two calcium ions bound in a central water filled tunnel. 496 water, 2 glycerol, 2 MES-buffer molecules, and 18 PEG fragments of different lengths could be refined in the solvent region. The 208 most reliable residues, without disorder or reduced occupancy in their side-chains, were finally refined without restraints. A subsequent full-matrix refinement cycle for the positional parameters yielded estimated standard deviations (esds) by matrix inversion. The herewith calculated bond lengths and bond-esds were used to obtain averaged bond lengths, which have been compared to the restraints used in preceding refinement cycles.

Acta Crystallographica Section D Biological Crystallography, 2003

The X-ray crystal structure of squid-type diisopropylfluorophosphatase (DFPase) has been refined ... more The X-ray crystal structure of squid-type diisopropylfluorophosphatase (DFPase) has been refined to a resolution of 0.85 A and a crystallographic R value of 9.4%. Crystal annealing improved both the mosaicity and resolution of the crystals considerably. The overall structure of this protein represents a six-bladed beta-propeller with two calcium ions bound in a central water-filled tunnel. 496 water, two glycerol and two MES buffer molecules and 18 PEG fragments of different lengths could be refined in the solvent region. 45 of the 314 residues have been refined with alternative orientations. H atoms have been omitted from disordered residues. For the residues of the inner beta-strands, H atoms are visible in a normal F(o) - F(c) difference map of a hydrogen-deficient structure model. The 208 most reliable residues, without disorder or reduced occupancy in their side chains, were finally refined without restraints. A subsequent full-matrix refinement cycle for the positional parameters yielded estimated standard deviations (e.s.d.s) by matrix inversion. The thus calculated bond lengths and bond angles and their e.s.d.s were used to obtain averaged bond lengths and bond angles, which were compared with the restraints applied in the preceding refinement cycles. The lengths and angles of the hydrogen bonds inside the antiparallel beta-sheets of the DFPase structure were compared with data averaged over 11 high-resolution protein structures. Torsion angles were averaged according to angle types used as restraints in X-PLOR and CNS and subsequently compared with values obtained from 46 high-resolution structures. Side-chain torsion angles were also classified into rotamer types according to the Penultimate Rotamer Library. Moreover, precise dimensions for both Ca(2+)-coordination polyhedra could be obtained and the coordination of one Ca(2+) ion by an imidazole N atom was confirmed. This statistical analysis thus provides a first step towards a set of restraints that are founded completely on macromolecular data; however, 10-20 additional protein data sets of comparable accuracy and size will be required to obtain a larger statistical base, especially for side-chain analysis.

Glucose-fructose oxidoreductase (E.C. 1. I .99.-) from the ethanol-producing Gram-negative bacter... more Glucose-fructose oxidoreductase (E.C. 1. I .99.-) from the ethanol-producing Gram-negative bacterium Zyrnomonas mobilis is a periplasmic, soluble enzyme that forms a homotetramer of 1 6 0 kDa with one NADP(H) cofactor per subunit that is tightly, but noncovalently, bound. The enzyme was crystallized by the hanging drop vapor diffusion method using sodium citrate as precipitant. The obtained crystals belong to the space group P21212, with unit cell constants of 84.6 A, 94.1 A, and 117.OA, consistent with two monomers in the asymmetric unit.