Elisabeth Hartmann
Publications
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3.42Impact points
A complete volume profile for the reversible binding of camphor to cytochrome P450(cam).
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 the reaction and activation volumes enabled the construction of the first complete volume profile for the r... [more] 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 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.
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3.28Impact points
Femtosecond free-electron laser x-ray diffraction data sets for algorithm development.
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 coherent diffraction methods freely available to the public, to bolster the development of algorithms that are ess... [more] 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 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.
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3.28Impact points
Unsupervised classification of single-particle X-ray diffraction snapshots by spectral clustering.
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 or more particles. Experimental data sets also often contain unintentional contamination with different s... [more] 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 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.
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34.48Impact points
Structure and mechanism of a bacterial light-regulated cyclic nucleotide phosphodiesterase.
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 in various proteins from the Bacteria, Euglenozoa and Fungi. Although structures of single-domain BLUF pr... [more] 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 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.
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3.87Impact points
On the structural basis of the catalytic mechanism and the regulation of the alpha subunit of tryptophan synthase from Salmonella typhimurium and BX1 from maize, two evolutionarily related enzymes.
Journal of molecular biology. 10/2005; 352(3):608-20.
Indole is a reaction intermediate in at least two biosynthetic pathways in maize seedlings. In the primary metabolism, the alpha-subunit (TSA) of the bifunctional tryptophan synthase (TRPS) catalyzes the cleavage of indole 3-glycerol phosphate (IGP) to indole and d-glyceraldehyde 3-phosphate (G3P). ... [more] Indole is a reaction intermediate in at least two biosynthetic pathways in maize seedlings. In the primary metabolism, the alpha-subunit (TSA) of the bifunctional tryptophan synthase (TRPS) catalyzes the cleavage of indole 3-glycerol phosphate (IGP) to indole and d-glyceraldehyde 3-phosphate (G3P). Subsequently, indole diffuses through the connecting tunnel to the beta-active site where it is condensed with serine to form tryptophan and water. The maize enzyme, BX1, a homolog of TSA, also cleaves IGP to G3P and indole, and the indole is further converted to 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one, a secondary plant metabolite. BX1 cleaves IGP significantly faster to G3P and indole than does TSA. In line with their different biological functions, these two evolutionary related enzymes differ significantly in their regulatory aspects while catalyzing the same chemistry. Here, the mechanism of IGP cleavage by TSA was analyzed using a novel transition state analogue generated in situ by reaction of 2-aminophenol and G3P. The crystal structure of the complex shows an sp3-hybridized atom corresponding to the C3 position of IGP. The catalytic alphaGlu49 rotates to interact with the sp3-hybridized atom and the 3' hydroxyl group suggesting that it serves both as proton donor and acceptor in the alpha-reaction. The second catalytic residue, alphaAsp60 interacts with the atom corresponding to the indolyl nitrogen, and the catalytically important loop alphaL6 is in the closed, high activity conformation. Comparison of the TSA and TSA-transition state analogue structures with the crystal structure of BX1 suggests that the faster catalytic rate of BX1 may be due to a stabilization of the active conformation: loop alphaL6 is closed and the catalytic glutamate is in the active conformation. The latter is caused by a substitution of the residues that stabilize the inactive conformation in TRPS.
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4.80Impact points
Structural analysis of isoform-specific inhibitors targeting the tetrahydrobiopterin binding site of human nitric oxide synthases.
Journal of medicinal chemistry. 08/2005; 48(15):4783-92.
Nitric oxide synthesized from l-arginine by nitric oxide synthase isoforms (NOS-I-III) is physiologically important but also can be deleterious when overproduced. Selective NOS inhibitors are of clinical interest, given their differing pathophysiological roles. Here we describe our approach to targe... [more] Nitric oxide synthesized from l-arginine by nitric oxide synthase isoforms (NOS-I-III) is physiologically important but also can be deleterious when overproduced. Selective NOS inhibitors are of clinical interest, given their differing pathophysiological roles. Here we describe our approach to target the unique NOS (6R,1'R,2'S)-5,6,7,8-tetrahydrobiopterin (H(4)Bip) binding site. By a combination of ligand- and structure-based design, the structure-activity relationship (SAR) for a focused set of 41 pteridine analogues on four scaffolds was developed, revealing selective NOS-I inhibitors. The X-ray crystal structure of rat NOS-I dimeric-oxygenase domain with H(4)Bip and l-arginine was determined and used for human isoform homology modeling. All available NOS structural information was subjected to comparative analysis of favorable protein-ligand interactions using the GRID/concensus principal component analysis (CPCA) approach to identify the isoform-specific interaction site. Our interpretation, based on protein structures, is in good agreement with the ligand SAR and thus permits the rational design of next-generation inhibitors targeting the H(4)Bip binding site with enhanced isoform selectivity for therapeutics in pathology with NO overproduction.
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5.33Impact points
Structural basis for the specificity of the nitric-oxide synthase inhibitors W1400 and Nomega-propyl-L-Arg for the inducible and neuronal isoforms.
The Journal of biological chemistry. 12/2003; 278(46):45818-25.
The high level of amino acid conservation and structural similarity in the immediate vicinity of the substrate binding sites of the oxygenase domains of the nitric-oxide synthase (NOS) isoforms (eNOSoxy, iNOSoxy, and nNOSoxy) make the interpretation of the structural basis of inhibitor isoform speci... [more] The high level of amino acid conservation and structural similarity in the immediate vicinity of the substrate binding sites of the oxygenase domains of the nitric-oxide synthase (NOS) isoforms (eNOSoxy, iNOSoxy, and nNOSoxy) make the interpretation of the structural basis of inhibitor isoform specificity a challenge and provide few clues for the design of new selective compounds. Crystal structures of iNOSoxy and nNOSoxy complexed with the inhibitors W1400 and Nomega-propyl-l-arginine provide a rationale for their isoform specificity. It involves differences outside the immediate active site as well as a conformational flexibility in the active site that allows the adoption of distinct conformations in response to interactions with the inhibitors. This flexibility is determined by isoform-specific residues outside the active site.
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4.39Impact points
Crystal structures and molecular mechanism of a light-induced signaling switch: The Phot-LOV1 domain from Chlamydomonas reinhardtii.
Biophysical journal. 05/2003; 84(4):2474-82.
Phot proteins (phototropins and homologs) are blue-light photoreceptors that control mechanical processes like phototropism, chloroplast relocation, or guard-cell opening in plants. Phot receptors consist of two flavin mononucleotide (FMN)-binding light, oxygen, or voltage (LOV) domains and a C-term... [more] Phot proteins (phototropins and homologs) are blue-light photoreceptors that control mechanical processes like phototropism, chloroplast relocation, or guard-cell opening in plants. Phot receptors consist of two flavin mononucleotide (FMN)-binding light, oxygen, or voltage (LOV) domains and a C-terminal serine/threonine kinase domain. We determined crystal structures of the LOV1 domain of Phot1 from the green alga Chlamydomonas reinhardtii in the dark and illuminated state to 1.9 A and 2.8 A resolution, respectively. The structure resembles that of LOV2 from Adiantum (Crosson, S. and K. Moffat. 2001. PROC: Natl. Acad. Sci. USA. 98:2995-3000). In the resting dark state of LOV1, the reactive Cys-57 is present in two conformations. Blue-light absorption causes formation of a proposed active signaling state that is characterized by a covalent bond between the flavin C4a and the thiol of Cys-57. There are differences around the FMN chromophore but no large overall conformational changes. Quantum chemical calculations based on the crystal structures revealed the electronic distribution in the active site during the photocycle. The results suggest trajectories for electrons, protons, and the active site cysteine and offer an interpretation of the reaction mechanism.
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On the Structural Basis of the Catalytic Mechanism and the Regulation of the Alpha Subunit of Tryptophan Synthase from Salmonella typhimurium and BX1 from Maize, Two Evolutionarily Related Enzymes
Journal of Molecular Biology.
Indole is a reaction intermediate in at least two biosynthetic pathways in maize seedlings. In the primary metabolism, the α-subunit (TSA) of the bifunctional tryptophan synthase (TRPS) catalyzes the cleavage of indole 3-glycerol phosphate (IGP) to indole and d-glyceraldehyde 3-phosphate (G3P). Subs... [more] Indole is a reaction intermediate in at least two biosynthetic pathways in maize seedlings. In the primary metabolism, the α-subunit (TSA) of the bifunctional tryptophan synthase (TRPS) catalyzes the cleavage of indole 3-glycerol phosphate (IGP) to indole and d-glyceraldehyde 3-phosphate (G3P). Subsequently, indole diffuses through the connecting tunnel to the β-active site where it is condensed with serine to form tryptophan and water. The maize enzyme, BX1, a homolog of TSA, also cleaves IGP to G3P and indole, and the indole is further converted to 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one, a secondary plant metabolite. BX1 cleaves IGP significantly faster to G3P and indole than does TSA. In line with their different biological functions, these two evolutionary related enzymes differ significantly in their regulatory aspects while catalyzing the same chemistry. Here, the mechanism of IGP cleavage by TSA was analyzed using a novel transition state analogue generated in situ by reaction of 2-aminophenol and G3P. The crystal structure of the complex shows an sp3-hybridized atom corresponding to the C3 position of IGP. The catalytic αGlu49 rotates to interact with the sp3-hybridized atom and the 3′ hydroxyl group suggesting that it serves both as proton donor and acceptor in the α-reaction. The second catalytic residue, αAsp60 interacts with the atom corresponding to the indolyl nitrogen, and the catalytically important loop αL6 is in the closed, high activity conformation. Comparison of the TSA and TSA-transition state analogue structures with the crystal structure of BX1 suggests that the faster catalytic rate of BX1 may be due to a stabilization of the active conformation: loop αL6 is closed and the catalytic glutamate is in the active conformation. The latter is caused by a substitution of the residues that stabilize the inactive conformation in TRPS.
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Proceedings of SPIE
Proceedings of SPIE0277786X;
Results of coherent diffractive imaging experiments performed with soft X-rays (1-2 keV) at the Linac Coherent Light Source are presented. Both organic and inorganic nano-sized objects were injected into the XFEL beam as an aerosol focused with an aerodynamic ...... [more] Results of coherent diffractive imaging experiments performed with soft X-rays (1-2 keV) at the Linac Coherent Light Source are presented. Both organic and inorganic nano-sized objects were injected into the XFEL beam as an aerosol focused with an aerodynamic ...
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Femtosecond free-electron laser x-ray diffraction data sets for algorithm development
20(4):4149-4158.
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 coherent diffraction methods freely available to the public, to bolster the development of algorithms that are ess... [more] 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 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.
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Single particle imaging with soft x-rays at the Linac Coherent Light Source
Proc. SPIE. 8078:807809-807809-9.
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Femtosecond free-electron laser x-ray diffraction data sets for algorithm development
Optics Express. 20(4):4149-4158.
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On the Structural Basis of the Catalytic Mechanism and the Regulation of the Alpha Subunit of Tryptophan Synthase from Salmonella typhimurium and BX1 from Maize, Two Evolutionarily Related Enzymes
Journal of Molecular Biology, v.352, 608-620 (2005).
Indole is a reaction intermediate in at least two biosynthetic pathways in maize seedlings. In the primary metabolism, the α−subunit (TSA) of the bifunctional tryptophan synthase (TRPS) catalyzes the cleavage of indole 3−glycerol phosphate (IGP) to indole and d−glyceraldehyde 3−phosphate (G3P). Subs... [more] Indole is a reaction intermediate in at least two biosynthetic pathways in maize seedlings. In the primary metabolism, the α−subunit (TSA) of the bifunctional tryptophan synthase (TRPS) catalyzes the cleavage of indole 3−glycerol phosphate (IGP) to indole and d−glyceraldehyde 3−phosphate (G3P). Subsequently, indole diffuses through the connecting tunnel to the β−active site where it is condensed with serine to form tryptophan and water. The maize enzyme, BX1, a homolog of TSA, also cleaves IGP to G3P and indole, and the indole is further converted to 2,4−dihydroxy−7−methoxy−2H−1,4−benzoxazin−3(4H)−one, a secondary plant metabolite. BX1 cleaves IGP significantly faster to G3P and indole than does TSA. In line with their different biological functions, these two evolutionary related enzymes differ significantly in their regulatory aspects while catalyzing the same chemistry. Here, the mechanism of IGP cleavage by TSA was analyzed using a novel transition state analogue generated in situ by reaction of 2−aminophenol and G3P. The crystal structure of the complex shows an sp3−hybridized atom corresponding to the C3 position of IGP. The catalytic αGlu49 rotates to interact with the sp3−hybridized atom and the 3′ hydroxyl group suggesting that it serves both as proton donor and acceptor in the α−reaction. The second catalytic residue, αAsp60 interacts with the atom corresponding to the indolyl nitrogen, and the catalytically important loop αL6 is in the closed, high activity conformation. Comparison of the TSA and TSA−transition state analogue structures with the crystal structure of BX1 suggests that the faster catalytic rate of BX1 may be due to a stabilization of the active conformation: loop αL6 is closed and the catalytic glutamate is in the active conformation. The latter is caused by a substitution of the residues that stabilize the inactive conformation in TRPS.
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Structure and mechanism of a bacterial light−regulated cyclic nucleotide phosphodiesterase
Nature, v.459, 1015-1018 (2009).
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 chromophore1. BLUF domains are present in various proteins from the Bacteria, Euglenozoa and Fungi. Although structures of single−domain BLUF p... [more] 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 chromophore1. BLUF domains are present in various proteins from the Bacteria, Euglenozoa and Fungi. Although structures of single−domain BLUF proteins have been determined2, 3, 4, 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 pneumoniae5. 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 Bacteria6, 7, 8, 9. 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.
Following (8)
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Max J Cryle
Max-Planck-Institut für medizinische Forschung -
Sasa Bajt
Deutsches Elektronen-Synchrotron -
Karol Nass
Deutsches Elektronen-Synchrotron -
Nicola Coppola
European XFEL GmbH -
Andreas Hartmann
Assistance Publique – Hôpitaux de Paris
Topics (3)
