Ilme Schlichting

Publications of Ilme Schlichting

• A complete volume profile for the reversible binding of camphor to cytochrome P450(cam).

  Authors: Alicja Franke, Elisabeth Hartmann, Ilme Schlichting, Rudi van Eldik

  Journal of biological inorganic chemistry : JBIC : a publication of the Society of Biological Inorganic Chemistry. 03/2012; 17(3):447-63.

  The effect of pressure on the kinetics and thermodynamics of the reversible binding of camphor to cytochrome P450(cam) was studied as a function of the K(+) concentration. The determination of theThe effect of pressure on the kinetics and thermodynamics of the reversible binding of camphor to cytochrome P450(cam) was studied as a function of the K(+) concentration. The determination of the reaction and activation volumes enabled the construction of the first complete volume profile for the reversible binding of camphor to P450(cam). Although the volume profiles constructed for the reactions conducted at low and high K(+) concentrations are rather similar, and both show a drastic volume increase on going from the reactant to the transition state and a relatively small volume change on going from the transition to the product state, the position of the transition state is largely affected by the K(+) concentration in solution. Similarly, the activation volume determined for the dissociation of camphor is influenced by the presence of K(+), which reflects changes in the ease of water entering the active site of camphor-bound P450(cam) that depends on the K(+) concentration. Careful analysis of the components that contribute to the observed volume changes allowed the estimation of the total number of water molecules expelled to the bulk solvent during the binding of camphor to P450(cam) and the subsequent spin transition. The results are discussed in reference to other studies reported in the literature that deal with the kinetics and thermodynamics of the binding of camphor to P450(cam) under various reaction conditions.

• Crystallography on a chip.

  Authors: Arash Zarrine-Afsar, Thomas R M Barends, Christina Müller, Martin R Fuchs, Lukas Lomb, Ilme Schlichting, R J Dwayne Miller

  Acta crystallographica. Section D, Biological crystallography. 03/2012; 68(Pt 3):321-3.

  A new chip-based crystal-mounting approach for rapid room-temperature data collection from numerous crystals is described. This work was motivated by the recent development of X-ray free-electronA new chip-based crystal-mounting approach for rapid room-temperature data collection from numerous crystals is described. This work was motivated by the recent development of X-ray free-electron lasers. These novel sources deliver very intense femtosecond X-ray pulses that promise to yield high-resolution diffraction data of nanocrystals before their destruction by radiation damage. Thus, the concept of `diffraction before destruction' requires rapid replenishment of the sample for each exposure. The chip promotes the self-assembly of an array of protein crystals on a surface. Rough features on the surface cause the crystals to adopt random orientations, allowing efficient sampling of reciprocal space.

• Femtosecond free-electron laser x-ray diffraction data sets for algorithm development.

  Authors: Stephan Kassemeyer, Jan Steinbrener, Lukas Lomb, Elisabeth Hartmann, Andrew Aquila, Anton Barty, Andrew V Martin, Christina Y Hampton, Saša Bajt, Miriam Barthelmess [......] Georg Weidenspointner, Uwe Weierstall, Thomas A White, Cornelia Wunderer, Matthias Frank, Henry N Chapman, Joachim Ullrich, Lothar Strüder, Michael J Bogan, Ilme Schlichting

  Optics express. 02/2012; 20(4):4149-58.

  We describe femtosecond X-ray diffraction data sets of viruses and nanoparticles collected at the Linac Coherent Light Source. The data establish the first large benchmark data sets for coherentWe describe femtosecond X-ray diffraction data sets of viruses and nanoparticles collected at the Linac Coherent Light Source. The data establish the first large benchmark data sets for coherent diffraction methods freely available to the public, to bolster the development of algorithms that are essential for developing this novel approach as a useful imaging technique. Applications are 2D reconstructions, orientation classification and finally 3D imaging by assembling 2D patterns into a 3D diffraction volume.

• Time-resolved protein nanocrystallography using an X-ray free-electron laser.

  Authors: Andrew Aquila, Mark S Hunter, R Bruce Doak, Richard A Kirian, Petra Fromme, Thomas A White, Jakob Andreasson, David Arnlund, Saša Bajt, Thomas R M Barends [......] Nicusor Timneanu, Joachim Ullrich, Xiaoyu Wang, Garth J Williams, Georg Weidenspointner, Uwe Weierstall, Cornelia Wunderer, Anton Barty, John C H Spence, Henry N Chapman

  Optics express. 01/2012; 20(3):2706-16.

  We demonstrate the use of an X-ray free electron laser synchronized with an optical pump laser to obtain X-ray diffraction snapshots from the photoactivated states of large membrane protein complexesWe demonstrate the use of an X-ray free electron laser synchronized with an optical pump laser to obtain X-ray diffraction snapshots from the photoactivated states of large membrane protein complexes in the form of nanocrystals flowing in a liquid jet. Light-induced changes of Photosystem I-Ferredoxin co-crystals were observed at time delays of 5 to 10 µs after excitation. The result correlates with the microsecond kinetics of electron transfer from Photosystem I to ferredoxin. The undocking process that follows the electron transfer leads to large rearrangements in the crystals that will terminally lead to the disintegration of the crystals. We describe the experimental setup and obtain the first time-resolved femtosecond serial X-ray crystallography results from an irreversible photo-chemical reaction at the Linac Coherent Light Source. This technique opens the door to time-resolved structural studies of reaction dynamics in biological systems.

• Lipidic phase membrane protein serial femtosecond crystallography.

  Authors: Linda C Johansson, David Arnlund, Thomas A White, Gergely Katona, Daniel P Deponte, Uwe Weierstall, R Bruce Doak, Robert L Shoeman, Lukas Lomb, Erik Malmerberg [......] Nicusor Timneanu, Joachim Ullrich, Weixiao Y Wahlgren, Xiaoyu Wang, Georg Weidenspointner, Cornelia Wunderer, Petra Fromme, Henry N Chapman, John C H Spence, Richard Neutze

  Nature methods. 01/2012; 9(3):263-5.

  X-ray free electron laser (X-FEL)-based serial femtosecond crystallography is an emerging method with potential to rapidly advance the challenging field of membrane protein structural biology. HereX-ray free electron laser (X-FEL)-based serial femtosecond crystallography is an emerging method with potential to rapidly advance the challenging field of membrane protein structural biology. Here we recorded interpretable diffraction data from micrometer-sized lipidic sponge phase crystals of the Blastochloris viridis photosynthetic reaction center delivered into an X-FEL beam using a sponge phase micro-jet.

• In vivo protein crystallization opens new routes in structural biology.

  Authors: Rudolf Koopmann, Karolina Cupelli, Lars Redecke, Karol Nass, Daniel P Deponte, Thomas A White, Francesco Stellato, Dirk Rehders, Mengning Liang, Jakob Andreasson [......] Georg Weidenspointner, Uwe Weierstall, Garth J Williams, Cornelia B Wunderer, Petra Fromme, John C H Spence, Thilo Stehle, Henry N Chapman, Christian Betzel, Michael Duszenko

  Nature methods. 01/2012; 9(3):259-62.

  Protein crystallization in cells has been observed several times in nature. However, owing to their small size these crystals have not yet been used for X-ray crystallographic analysis. We preparedProtein crystallization in cells has been observed several times in nature. However, owing to their small size these crystals have not yet been used for X-ray crystallographic analysis. We prepared nano-sized in vivo-grown crystals of Trypanosoma brucei enzymes and applied the emerging method of free-electron laser-based serial femtosecond crystallography to record interpretable diffraction data. This combined approach will open new opportunities in structural systems biology.

• Evolution of a new enzyme for carbon disulphide conversion by an acidothermophilic archaeon.

  Authors: Marjan J Smeulders, Thomas R M Barends, Arjan Pol, Anna Scherer, Marcel H Zandvoort, Anikó Udvarhelyi, Ahmad F Khadem, Andreas Menzel, John Hermans, Robert L Shoeman, Hans J C T Wessels, Lambert P van den Heuvel, Lina Russ, Ilme Schlichting, Mike S M Jetten, Huub J M Op den Camp

  Nature. 10/2011; 478(7369):412-6.

  Extremophilic organisms require specialized enzymes for their exotic metabolisms. Acid-loving thermophilic Archaea that live in the mudpots of volcanic solfataras obtain their energy from reducedExtremophilic organisms require specialized enzymes for their exotic metabolisms. Acid-loving thermophilic Archaea that live in the mudpots of volcanic solfataras obtain their energy from reduced sulphur compounds such as hydrogen sulphide (H(2)S) and carbon disulphide (CS(2)). The oxidation of these compounds into sulphuric acid creates the extremely acidic environment that characterizes solfataras. The hyperthermophilic Acidianus strain A1-3, which was isolated from the fumarolic, ancient sauna building at the Solfatara volcano (Naples, Italy), was shown to rapidly convert CS(2) into H(2)S and carbon dioxide (CO(2)), but nothing has been known about the modes of action and the evolution of the enzyme(s) involved. Here we describe the structure, the proposed mechanism and evolution of a CS(2) hydrolase from Acidianus A1-3. The enzyme monomer displays a typical β-carbonic anhydrase fold and active site, yet CO(2) is not one of its substrates. Owing to large carboxy- and amino-terminal arms, an unusual hexadecameric catenane oligomer has evolved. This structure results in the blocking of the entrance to the active site that is found in canonical β-carbonic anhydrases and the formation of a single 15-Å-long, highly hydrophobic tunnel that functions as a specificity filter. The tunnel determines the enzyme's substrate specificity for CS(2), which is hydrophobic. The transposon sequences that surround the gene encoding this CS(2) hydrolase point to horizontal gene transfer as a mechanism for its acquisition during evolution. Our results show how the ancient β-carbonic anhydrase, which is central to global carbon metabolism, was transformed by divergent evolution into a crucial enzyme in CS(2) metabolism.

• Unsupervised classification of single-particle X-ray diffraction snapshots by spectral clustering.

  Authors: Chun Hong Yoon, Peter Schwander, Chantal Abergel, Inger Andersson, Jakob Andreasson, Andrew Aquila, Saša Bajt, Miriam Barthelmess, Anton Barty, Michael J Bogan [......] Dmitri Starodub, Jan Steinbrener, Gunter Stier, Lothar Strüder, Martin Svenda, Joachim Ullrich, Georg Weidenspointner, Thomas A White, Cornelia Wunderer, Abbas Ourmazd

  Optics express. 08/2011; 19(17):16542-9.

  Single-particle experiments using X-ray Free Electron Lasers produce more than 10(5) snapshots per hour, consisting of an admixture of blank shots (no particle intercepted), and exposures of one orSingle-particle experiments using X-ray Free Electron Lasers produce more than 10(5) snapshots per hour, consisting of an admixture of blank shots (no particle intercepted), and exposures of one or more particles. Experimental data sets also often contain unintentional contamination with different species. We present an unsupervised method able to sort experimental snapshots without recourse to templates, specific noise models, or user-directed learning. The results show 90% agreement with manual classification.

• Femtosecond X-ray protein nanocrystallography.

  Authors: Henry N Chapman, Petra Fromme, Anton Barty, Thomas A White, Richard A Kirian, Andrew Aquila, Mark S Hunter, Joachim Schulz, Daniel P DePonte, Uwe Weierstall [......] Sebastian Schorb, Daniela Rupp, Marcus Adolph, Tais Gorkhover, Inger Andersson, Helmut Hirsemann, Guillaume Potdevin, Heinz Graafsma, Björn Nilsson, John C H Spence

  Nature. 02/2011; 470(7332):73-7.

  X-ray crystallography provides the vast majority of macromolecular structures, but the success of the method relies on growing crystals of sufficient size. In conventional measurements, the necessaryX-ray crystallography provides the vast majority of macromolecular structures, but the success of the method relies on growing crystals of sufficient size. In conventional measurements, the necessary increase in X-ray dose to record data from crystals that are too small leads to extensive damage before a diffraction signal can be recorded. It is particularly challenging to obtain large, well-diffracting crystals of membrane proteins, for which fewer than 300 unique structures have been determined despite their importance in all living cells. Here we present a method for structure determination where single-crystal X-ray diffraction 'snapshots' are collected from a fully hydrated stream of nanocrystals using femtosecond pulses from a hard-X-ray free-electron laser, the Linac Coherent Light Source. We prove this concept with nanocrystals of photosystem I, one of the largest membrane protein complexes. More than 3,000,000 diffraction patterns were collected in this study, and a three-dimensional data set was assembled from individual photosystem I nanocrystals (∼200 nm to 2 μm in size). We mitigate the problem of radiation damage in crystallography by using pulses briefer than the timescale of most damage processes. This offers a new approach to structure determination of macromolecules that do not yield crystals of sufficient size for studies using conventional radiation sources or are particularly sensitive to radiation damage.

• Single mimivirus particles intercepted and imaged with an X-ray laser.

  Authors: M Marvin Seibert, Tomas Ekeberg, Filipe R N C Maia, Martin Svenda, Jakob Andreasson, Olof Jönsson, Duško Odić, Bianca Iwan, Andrea Rocker, Daniel Westphal [......] Sebastian Schorb, Daniela Rupp, Marcus Adolph, Tais Gorkhover, Helmut Hirsemann, Guillaume Potdevin, Heinz Graafsma, Björn Nilsson, Henry N Chapman, Janos Hajdu

  Nature. 02/2011; 470(7332):78-81.

  X-ray lasers offer new capabilities in understanding the structure of biological systems, complex materials and matter under extreme conditions. Very short and extremely bright, coherent X-ray pulsesX-ray lasers offer new capabilities in understanding the structure of biological systems, complex materials and matter under extreme conditions. Very short and extremely bright, coherent X-ray pulses can be used to outrun key damage processes and obtain a single diffraction pattern from a large macromolecule, a virus or a cell before the sample explodes and turns into plasma. The continuous diffraction pattern of non-crystalline objects permits oversampling and direct phase retrieval. Here we show that high-quality diffraction data can be obtained with a single X-ray pulse from a non-crystalline biological sample, a single mimivirus particle, which was injected into the pulsed beam of a hard-X-ray free-electron laser, the Linac Coherent Light Source. Calculations indicate that the energy deposited into the virus by the pulse heated the particle to over 100,000 K after the pulse had left the sample. The reconstructed exit wavefront (image) yielded 32-nm full-period resolution in a single exposure and showed no measurable damage. The reconstruction indicates inhomogeneous arrangement of dense material inside the virion. We expect that significantly higher resolutions will be achieved in such experiments with shorter and brighter photon pulses focused to a smaller area. The resolution in such experiments can be further extended for samples available in multiple identical copies.

• X-ray and NMR crystallography in an enzyme active site: the indoline quinonoid intermediate in tryptophan synthase.

  Authors: Jinfeng Lai, Dimitri Niks, Yachong Wang, Tatiana Domratcheva, Thomas R M Barends, Friedrich Schwarz, Ryan A Olsen, Douglas W Elliott, M Qaiser Fatmi, Chia-en A Chang, Ilme Schlichting, Michael F Dunn, Leonard J Mueller

  Journal of the American Chemical Society. 01/2011; 133(1):4-7.

  Chemical-level details such as protonation and hybridization state are critical for understanding enzyme mechanism and function. Even at high resolution, these details are difficult to determine byChemical-level details such as protonation and hybridization state are critical for understanding enzyme mechanism and function. Even at high resolution, these details are difficult to determine by X-ray crystallography alone. The chemical shift in NMR spectroscopy, however, is an extremely sensitive probe of the chemical environment, making solid-state NMR spectroscopy and X-ray crystallography a powerful combination for defining chemically detailed three-dimensional structures. Here we adopted this combined approach to determine the chemically rich crystal structure of the indoline quinonoid intermediate in the pyridoxal-5'-phosphate-dependent enzyme tryptophan synthase under conditions of active catalysis. Models of the active site were developed using a synergistic approach in which the structure of this reactive substrate analogue was optimized using ab initio computational chemistry in the presence of side-chain residues fixed at their crystallographically determined coordinates. Various models of charge and protonation state for the substrate and nearby catalytic residues could be uniquely distinguished by their calculated effects on the chemical shifts measured at specifically (13)C- and (15)N-labeled positions on the substrate. Our model suggests the importance of an equilibrium between tautomeric forms of the substrate, with the protonation state of the major isomer directing the next catalytic step.

• Computational characterization of reaction intermediates in the photocycle of the sensory domain of the AppA blue light photoreceptor.

  Authors: Maria G Khrenova, Tatiana Domratcheva, Ilme Schlichting, Bella L Grigorenko, Alexander V Nemukhin

  Photochemistry and photobiology. 11/2010; 87(3):564-73.

  The AppA protein with the BLUF (blue light using flavin adenine dinucleotide) domain is a blue light photoreceptor that cycle between dark-adapted and light-induced functional states. WeThe AppA protein with the BLUF (blue light using flavin adenine dinucleotide) domain is a blue light photoreceptor that cycle between dark-adapted and light-induced functional states. We characterized possible reaction intermediates in the photocycle of AppA BLUF. Molecular dynamics (MD), quantum chemical and quantum mechanical-molecular mechanical (QM/MM) calculations were carried out to describe several stable structures of a molecular system modeling the protein. The coordinates of heavy atoms from the crystal structure (PDB code 2IYG) of the protein in the dark state served as starting point for 10 ns MD simulations. Representative MD frames were used in QM(B3LYP/cc-pVDZ)/MM(AMBER) calculations to locate minimum energy configurations of the model system. Vertical electronic excitation energies were estimated for the molecular clusters comprising the quantum subsystems of the QM/MM optimized structures using the SOS-CIS(D) quantum chemistry method. Computational results support the occurrence of photoreaction intermediates that are characterized by spectral absorption bands between those of the dark and light states. They agree with crystal structures of reaction intermediates (PDB code 2IYI) observed in the AppA BLUF domain. Transformations of the Gln63 side chain stimulated by photo-excitation and performed with the assistance of the chromophore and the Met106 side chain are responsible for these intermediates.

• Structural characterization of CYP165D3, a cytochrome P450 involved in phenolic coupling in teicoplanin biosynthesis.

  Authors: Max J Cryle, Jessica Staaden, Ilme Schlichting

  Archives of biochemistry and biophysics. 10/2010; 507(1):163-73.

  Teicoplanin is a glycopeptide antibiotic with activity against Gram-positive bacteria and remains one of the last lines of clinical defense against certain bacterial infections. We have cloned,Teicoplanin is a glycopeptide antibiotic with activity against Gram-positive bacteria and remains one of the last lines of clinical defense against certain bacterial infections. We have cloned, expressed, and purified the cytochrome P450 OxyE (CYP165D3) from the teicoplanin biosynthetic gene cluster of Actinoplanes teichomyceticus, which is responsible for the phenolic coupling of the aromatic side chains of the first and third peptide residues in the teicoplanin peptide. The crystal structure of OxyE has been determined to 2.5Å resolution, revealing the probable binding surface for the carrier protein substrate and an extension of the active site into a pocket located above the β-1 sheet. The binding of potential substrates to OxyE shows that peptidyl carrier protein-bound linear peptides bind to OxyE, albeit with low affinity in the absence of a phenolic cross-link that should normally be installed by another Oxy protein in the teicoplanin biosynthetic pathway. This result indicates that the carrier protein alone is not sufficient for tight substrate binding to OxyE and that the Oxy proteins sense the structure of the bound peptide in addition to the presence of the carrier protein, a feature distinct from other carrier protein/P450 systems.

• Structural and biochemical characterization of the cytochrome P450 CypX (CYP134A1) from Bacillus subtilis: a cyclo-L-leucyl-L-leucyl dipeptide oxidase.

  Authors: Max J Cryle, Stephen G Bell, Ilme Schlichting

  Biochemistry. 08/2010; 49(34):7282-96.

  Cytochrome P450 CypX (CYP134A1), isolated from Bacillus subtilis, has previously been implicated in the three-step oxidative transformation of the diketopiperazine cyclo-l-leucyl-l-leucyl intoCytochrome P450 CypX (CYP134A1), isolated from Bacillus subtilis, has previously been implicated in the three-step oxidative transformation of the diketopiperazine cyclo-l-leucyl-l-leucyl into pulcherriminic acid, a precursor of the extracellular iron chelate pulcherrimin. In this study, we present the first experimental data relating to CYP134A1, where we show that CYP134A1 binds cyclo-l-leucyl-l-leucyl with an affinity of 24.5 +/- 0.5 muM. Structurally related diketopiperazines sharing similar alkyl side chains to cyclo-l-leucyl-l-leucyl also bind to CYP134A1 with comparable affinity. CYP134A1 is capable of catalyzing the in vitro oxidation of diketopiperazine substrates when supported with several alternate electron transfer partner systems. Products containing one additional oxygen atom and which are intermediate products of the expected pulcherriminic acid were identified by GCMS. The oxidation of related diketopiperazines reveals that different oxidative pathways exist for CYP134A1-catalyzed diketopiperazine oxidation. The crystal structure of CYP134A1 has been determined to 2.7 A resolution in the absence of substrate and in the presence of bound phenylimidazole ligands to 3.1 and 3.2 A resolution. The active site is dominated by hydrophobic residues and contains an unusual proline residue in place of the normally conserved alcohol residue that typically plays an important role in oxygen activation. The B-B(2) substrate recognition loop, which forms part of the active site, shows considerable flexibility and was found in both open and closed conformations in different structures. These results represent the first insights into the structural and biochemical basis underlying the multistep oxidation catalyzed by CYP134A1.

• Structural characterization of OxyD, a cytochrome P450 involved in beta-hydroxytyrosine formation in vancomycin biosynthesis.

  Authors: Max J Cryle, Anton Meinhart, Ilme Schlichting

  The Journal of biological chemistry. 08/2010; 285(32):24562-74.

  The cytochrome P450 OxyD from the balhimycin glycopeptide antibiotic biosynthetic operon of Amycolatopsis mediterranei is involved in the biosynthesis of the modified amino acidThe cytochrome P450 OxyD from the balhimycin glycopeptide antibiotic biosynthetic operon of Amycolatopsis mediterranei is involved in the biosynthesis of the modified amino acid beta-R-hydroxytyrosine, an essential precursor for biosynthesis of the vancomycin-type aglycone. OxyD binds the substrate tyrosine not free in solution, but rather covalently linked to the carrier protein (CP) domain of the non-ribosomal peptide synthase BpsD, exhibiting micromolar binding affinity to a tyrosine-loaded carrier protein construct. The crystal structure of OxyD was determined to 2.1-A resolution, revealing a potential binding site for the carrier protein-bound substrate in a different orientation to that seen with the acyl carrier protein-bound P450(BioI) (Cryle, M. J., and Schlichting, I. (2008) Proc. Natl. Acad. Sci. U.S.A. 105, 15696-15701). A series of residues were identified across known aminoacyl-CP-oxidizing P450s that are highly conserved and cluster in the active site or potential CP binding site of OxyD. These residues appear to be characteristic for aminoacyl-CP-oxidizing P450s, allowing sequence based identification of P450 function for this subgroup of P450s that play vital roles in the biosyntheses of many important natural products in addition to the vancomycin-type antibiotics. The ability to analyze such P450 function based upon sequence data alone should prove an important tool in the analysis and identification of new medicinally relevant biomolecules.

• Electronic Structure of (6-4) DNA Photoproduct Repair Involving a Non-Oxetane Pathway.

  Authors: Tatiana Domratcheva, Ilme Schlichting

  Journal of the American Chemical Society. 11/2009;

  Mutagenic pyrimidine-pyrimidone (6-4) photoproducts are one of the main DNA lesions induced by solar UV radiation. These lesions can be photoreversed by (6-4) photolyases. The originally publishedMutagenic pyrimidine-pyrimidone (6-4) photoproducts are one of the main DNA lesions induced by solar UV radiation. These lesions can be photoreversed by (6-4) photolyases. The originally published repair mechanism involves rearrangement of the lesion into an oxetane intermediate upon binding to the (6-4) photolyase, followed by light-induced electron transfer from the reduced flavin cofactor. In a recent crystallographic study on a (6-4) photoproduct complexed with (6-4) photolyase from Drosophila melanogaster no oxetane was observed, raising the possibility of a non-oxetane repair mechanism. Using quantum-chemical calculations we find that in addition to repair via an oxetane, a direct transfer of the hydroxyl group results in reversal of the radical anion (6-4) photoproduct. In both mechanisms, the transition states have high energies and correspond to avoided crossings of the ground and excited electronic states. To study whether the repair can proceed via these state crossings, the excited-state potential energy curves were computed. The radical excitation energies and accessibility of the nonadiabatic repair path were found to depend on hydrogen bonds and the protonation state of the lesion. On the basis of the energy calculations, a nonadiabatic repair of the excited (6-4) lesion radical anion via hydroxyl transfer is probable. This repair mechanism is in line with the recent structural data on the (6-4) photolyase from D. melanogaster .

• Tryptophan Synthase: Structure and Function of the Monovalent Cation Site.

  Authors: Adam T Dierkers, Dimitri Niks, Ilme Schlichting, Michael F Dunn

  Biochemistry. 10/2009;

  The monovalent cation (MVC) site of the tryptophan synthase from Salmonella typhimurium plays essential roles in catalysis and in the regulation of substrate channeling. In vitro, MVCs affect theThe monovalent cation (MVC) site of the tryptophan synthase from Salmonella typhimurium plays essential roles in catalysis and in the regulation of substrate channeling. In vitro, MVCs affect the equilibrium distribution of intermediates formed in the reaction of l-Ser with the alpha(2)beta(2) complex; the MVC-free, Cs(+)-bound, and NH(4)(+)-bound enzymes stabilize the alpha-aminoacrylate species, E(A-A), while Na(+) binding stabilizes the l-Ser external aldimine species, E(Aex(1)). Two probes of beta-site reactivity and conformation were used herein, the reactive indole analogue, indoline, and the l-Trp analogue, l-His. MVC-bound E(A-A) reacts rapidly with indoline to give the indoline quinonoid species, E(Q)(indoline), which slowly converts to dihydroiso-l-tryptophan. MVC-free E(A-A) gives very little E(Q)(indoline), and turnover is strongly impaired; the fraction of E(Q)(indoline) formed is <3.5% of that given by the Na(+)-bound form. The reaction of l-Ser with the MVC-free internal aldimine species, E(Ain), initially gives small amounts of an active E(A-A) which converts to an inactive species on a slower, conformational, time scale. This inactivation is abolished by the binding of MVCs. The inactive E(A-A) appears to have a closed beta-subunit conformation with an altered substrate binding site that is different from the known conformations of tryptophan synthase. Reaction of l-His with E(Ain) gives an equilibrating mixture of external aldimine and quinonoid species, E(Aex)(his) and E(Q)(his). The MVC-free and Na(+) forms of the enzyme gave trace amounts of E(Q)(his) ( approximately 1% of the beta-sites). The Cs(+) and NH(4)(+) forms gave approximately 17 and approximately 14%, respectively. The reactivity of MVC-free E(Ain) was restored by the binding of an alpha-site ligand. These studies show MVCs and alpha-site ligands act synergistically to modulate the switching of the beta-subunit from the open to the closed conformation, and this switching is crucial to the regulation of beta-site catalytic activity. Comparison of the structures of Na(+) and Cs(+) forms of the enzyme shows Cs(+) favors complexes with open indole binding sites poised for the conformational transition to the closed state, whereas the Na(+) form does not. The beta-subunits of Cs(+) complexes exhibit preformed indole subsites; the indole subsites of the open Na(+) complexes are collapsed, distorted, and too small to accommodate indole.

• Structure and mechanism of a bacterial light-regulated cyclic nucleotide phosphodiesterase.

  Authors: Thomas R M Barends, Elisabeth Hartmann, Julia J Griese, Thorsten Beitlich, Natalia V. Kirienko, Dmitri A Ryjenkov, Jochen Reinstein, Robert L Shoeman, Mark Gomelsky, Ilme Schlichting

  Nature. 07/2009; 459(7249):1015-8.

  The ability to respond to light is crucial for most organisms. BLUF is a recently identified photoreceptor protein domain that senses blue light using a FAD chromophore. BLUF domains are present inThe ability to respond to light is crucial for most organisms. BLUF is a recently identified photoreceptor protein domain that senses blue light using a FAD chromophore. BLUF domains are present in various proteins from the Bacteria, Euglenozoa and Fungi. Although structures of single-domain BLUF proteins have been determined, none are available for a BLUF protein containing a functional output domain; the mechanism of light activation in this new class of photoreceptors has thus remained poorly understood. Here we report the biochemical, structural and mechanistic characterization of a full-length, active photoreceptor, BlrP1 (also known as KPN_01598), from Klebsiella pneumoniae. BlrP1 consists of a BLUF sensor domain and a phosphodiesterase EAL output domain which hydrolyses cyclic dimeric GMP (c-di-GMP). This ubiquitous second messenger controls motility, biofilm formation, virulence and antibiotic resistance in the Bacteria. Crystal structures of BlrP1 complexed with its substrate and metal ions involved in catalysis or in enzyme inhibition provide a detailed understanding of the mechanism of the EAL-domain c-di-GMP phosphodiesterases. These structures also sketch out a path of light activation of the phosphodiesterase output activity. Photon absorption by the BLUF domain of one subunit of the antiparallel BlrP1 homodimer activates the EAL domain of the second subunit through allosteric communication transmitted through conserved domain-domain interfaces.

• Probing the role of the proximal heme ligand in cytochrome P450cam by recombinant incorporation of selenocysteine.

  Authors: Caroline Aldag, Igor A Gromov, Inés García-Rubio, Konstanze von Koenig, Ilme Schlichting, Bernhard Jaun, Donald Hilvert

  Proceedings of the National Academy of Sciences of the United States of America. 05/2009; 106(14):5481-6.

  The unique monooxygenase activity of cytochrome P450cam has been attributed to coordination of a cysteine thiolate to the heme cofactor. To investigate this interaction, we replaced cysteine with theThe unique monooxygenase activity of cytochrome P450cam has been attributed to coordination of a cysteine thiolate to the heme cofactor. To investigate this interaction, we replaced cysteine with the more electron-donating selenocysteine. Good yields of the selenoenzyme were obtained by bacterial expression of an engineered gene containing the requisite UGA codon for selenocysteine and a simplified yet functional selenocysteine insertion sequence (SECIS). The sulfur-to-selenium substitution subtly modulates the structural, electronic, and catalytic properties of the enzyme. Catalytic activity decreases only 2-fold, whereas substrate oxidation becomes partially uncoupled from electron transfer, implying a more complex role for the axial ligand than generally assumed.

• A preliminary neutron diffraction study of gamma-chymotrypsin.

  Authors: Walter R P Novak, Aaron G Moulin, Matthew P. Blakeley, Ilme Schlichting, Gregory A Petsko, Dagmar Ringe

  Acta crystallographica. Section F, Structural biology and crystallization communications. 04/2009; 65(Pt 3):317-20.

  The crystal preparation and preliminary neutron diffraction analysis of gamma-chymotrypsin are presented. Large hydrogenated crystals of gamma-chymotrypsin were exchanged into deuterated buffer viaThe crystal preparation and preliminary neutron diffraction analysis of gamma-chymotrypsin are presented. Large hydrogenated crystals of gamma-chymotrypsin were exchanged into deuterated buffer via vapor diffusion in a capillary and neutron Laue diffraction data were collected from the resulting crystal to 2.0 A resolution on the LADI-III diffractometer at the Institut Laue-Langevin (ILL) at room temperature. The neutron structure of a well studied protein such as gamma-chymotrypsin, which is also amenable to ultrahigh-resolution X-ray crystallography, represents the first step in developing a model system for the study of H atoms in protein crystals.