FIP: a highly automated beamline for multiwavelength anomalous diffraction experiments

Roth, M.; Carpentier, Philippe; Kaïkati, O.; Joly, J.; Charrault, P.; Pirocchi, Michel; Kahn, Richard; Fanchon, Éric; Jacquamet, Lilian; Borel, Franck; Bertoni, A.; Israel-Gouy, P.; Ferrer, Jean‐Luc

doi:10.1107/s0907444902003943

Cited by 81 publications

(71 citation statements)

References 16 publications

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“…For data collection, the crystals were frozen in liquid nitrogen after soaking in cryoprotectant buffer containing 10% polyethylene glycol 3000, 30% polyethylene glycol 400, and 0.1 M Tris-HCl, pH 8.5. Crystals of the native and Se-Met labeled proteins diffracted to 1.9 and 2.3 Å, respectively (beamline BM30A at the European Synchrotron Radiation Facility, Grenoble, France) (12).…”

Section: Methodsmentioning

confidence: 99%

Crystal Structure and Functional Characterization of Yeast YLR011wp, an Enzyme with NAD(P)H-FMN and Ferric Iron Reductase Activities

Liger

Graille

Zhou

et al. 2004

Journal of Biological Chemistry

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Flavodoxins are involved in a variety of electron transfer reactions that are essential for life. Although FMN-binding proteins are well characterized in prokaryotic organisms, information is scarce for eukaryotic flavodoxins. We describe the 2.0-Å resolution crystal structure of the Saccharomyces cerevisiae YLR011w gene product, a predicted flavoprotein. YLR011wp indeed adopts a flavodoxin fold, binds the FMN cofactor, and self-associates as a homodimer. Despite the absence of the flavodoxin key fingerprint motif involved in FMN binding, YLR011wp binds this cofactor in a manner very analogous to classical flavodoxins. YLR011wp closest structural homologue is the homodimeric Bacillus subtilis Yhda protein (25% sequence identity) whose homodimer perfectly superimposes onto the YLR011wp one. Yhda, whose function is not documented, has 53% sequence identity with the Bacillus sp. OY1-2 azoreductase. We show that YLR011wp has an NAD(P)H-dependent FMN reductase and a strong ferricyanide reductase activity. We further demonstrate a weak but specific reductive activity on azo dyes and nitrocompounds.The most frequently used cofactors in enzymatic redox reactions are the pyridine (NAD and NADP) and the flavin (FAD and FMN) nucleotides. Although NAD and NADP are soluble cofactors used by dehydrogenases, FAD and FMN usually work as prosthetic groups in flavoproteins to which they are tightly bound. Flavodoxins are small monomeric flavoproteins (15-22 kDa) that noncovalently bind a single FMN molecule, acting as a redox center (1). The flavodoxin scaffold contributes to the mechanism of electron transfer by stabilizing the FMN molecule in an environment that promotes the highly negative redox potential required for its biochemical activity. This is accomplished by sandwiching the FMN ring between two hydrophobic side chains. Flavodoxins are involved in a variety of electron transfer reactions that are essential in the metabolism of pyruvate (2), nitrogen, and pyridine nucleotides (3). These enzymes exist under three redox states: oxidized; partially reduced (semiquinone); and fully reduced (hydroquinone). Until now, flavodoxins have been identified in many prokaryotes and also in some eukaryotic algae (4 -6). In mammalian systems, flavodoxins have only been found as domains inserted in larger redox proteins such as cytochrome P450 reductase where they act as the electron transport intermediate between FAD and the P450 iron center (7). Flavodoxin-like domains are also found in mammalian nitric-oxide synthase (8) and in human erythrocyte NADPH-flavin reductase (9).The Saccharomyces cerevisiae YLR011w open reading frame codes for a protein of unknown function that has been found to be up-regulated in response to low temperature exposure (10). The sequence belongs to a COG4530 (Cluster of Orthologous Genes) predicted to generally contain flavoproteins. Hence, the YLR011wp 21-kDa protein was proposed to be composed of a single domain present in NADPH-dependent FMN reductases. We have solved the 2-Å resolution crystal structure, ...

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Section: Methodsmentioning

confidence: 99%

Crystal Structure and Functional Characterization of Yeast YLR011wp, an Enzyme with NAD(P)H-FMN and Ferric Iron Reductase Activities

Liger

Graille

Zhou

et al. 2004

Journal of Biological Chemistry

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“…Good diffracting crystals were obtained for mBIO3-BIO1/KAPA and mBIO3-BIO1/ADP/DTB using 0.2 M ammonium tartrate, pH 6.2, and 15% (w/v) polyethylene glycol 3350. Data for the native protein, [SeMet]-mBIO3-BIO1, and mBIO3-BIO1/KAPA were collected on the FIP-BM30A beamline (Roth et al, 2002) at the European Synchrotron Radiation Facility (Grenoble, France) using an ADSC Q315r charge-coupled device detector. Data for mBIO3-BIO1/ ADP/DTB were collected on ID14-1 at the European Synchrotron Radiation Facility using an ADSC Q210 charge-coupled device detector.…”

Section: Crystallization and Data Collectionmentioning

confidence: 99%

Biochemical and Structural Characterization of the Arabidopsis Bifunctional Enzyme Dethiobiotin Synthetase–Diaminopelargonic Acid Aminotransferase: Evidence for Substrate Channeling in Biotin Synthesis

et al. 2012

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Diaminopelargonic acid aminotransferase (DAPA-AT) and dethiobiotin synthetase (DTBS) catalyze the antepenultimate and the penultimate steps, respectively, of biotin synthesis. Whereas DAPA-AT and DTBS are encoded by distinct genes in bacteria, in biotin-synthesizing eukaryotes (plants and most fungi), both activities are carried out by a single enzyme encoded by a bifunctional gene originating from the fusion of prokaryotic monofunctional ancestor genes. In few angiosperms, including Arabidopsis thaliana, this chimeric gene (named BIO3-BIO1) also produces a bicistronic transcript potentially encoding separate monofunctional proteins that can be produced following an alternative splicing mechanism. The functional significance of the occurrence of a bifunctional enzyme in biotin synthesis pathway in eukaryotes and the relative implication of each of the potential enzyme forms (bifunctional versus monofunctional) in the plant biotin pathway are unknown. In this study, we demonstrate that the BIO3-BIO1 fusion protein is the sole protein form produced by the BIO3-BIO1 locus in Arabidopsis. The enzyme catalyzes both DAPA-AT and DTBS reactions in vitro and is targeted to mitochondria in vivo. Our biochemical and kinetic characterizations of the pure recombinant enzyme show that in the course of the reaction, the DAPA intermediate is directly transferred from the DAPA-AT active site to the DTBS active site. Analysis of several structures of the enzyme crystallized in complex with and without its ligands reveals key structural elements involved for acquisition of bifunctionality and brings, together with mutagenesis experiments, additional evidences for substrate channeling.

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“…Data were collected at the Institut Biologie Physique et Chimie (IBPC, Paris, France) by use of the generator RIGAKU model Micro7 (to 2.1 Å) and on the beamline FIP-BM30A (to 1.6 Å for KPC-2 and 1.23 Å for the C-ter-deleted mutant) at the ESRF (European Synchrotron Radiation Facility, Grenoble, France) (48). These data were processed with XDS (25).…”

Section: Vol 52 2008 Genetic and Structural Analysis Of Kpc-2 3727mentioning

confidence: 99%

Genetic and Structural Insights into the Dissemination Potential of the Extremely Broad-Spectrum Class A β-Lactamase KPC-2 Identified in an Escherichia coli Strain and an Enterobacter cloacae Strain Isolated from the Same Patient in France

Pétrella

Ziental-Gelus

Mayer

et al. 2008

Antimicrob Agents Chemother

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Two clinical strains of Escherichia coli (2138) and Enterobacter cloacae (7506) isolated from the same patient in France and showing resistance to extended-spectrum cephalosporins and low susceptibility to imipenem were investigated. Both strains harbored the plasmid-contained bla TEM-1 and bla KPC-2 genes. bla KLUC-2 , encoding a mutant of the chromosomal ␤-lactamase of Kluyvera cryocrescens, was also identified at a plasmid location in E. cloacae 7506, suggesting the ISEcp1-assisted escape of bla KLUC from the chromosome. Determination of the KPC-2 structure at 1.6 Å revealed that the binding site was occupied by the C-terminal (C-ter) residues coming from a symmetric KPC-2 monomer, with the ultimate C-ter Glu interacting with Ser130, Lys234, Thr235, and Thr237 in the active site. This mode of binding can be paralleled to the inhibition of the TEM-1 ␤-lactamase by the inhibitory protein BLIP. Determination of the 1.23-Å structure of a KPC-2 mutant in which the five C-ter residues were deleted revealed that the catalytic site was filled by a citrate molecule. Structure analysis and docking simulations with cefotaxime and imipenem provided further insights into the molecular basis of the extremely broad spectrum of KPC-2, which behaves as a cefotaximase with significant activity against carbapenems. In particular, residues 104, 105, 132, and 167 draw a binding cavity capable of accommodating both the aminothiazole moiety of cefotaxime and the 6␣-hydroxyethyl group of imipenem, with the binding of the former drug being also favored by a significant degree of freedom at the level of the loop at positions 96 to 105 and by an enlargement of the binding site at the end of strand ␤3.

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FIP: a highly automated beamline for multiwavelength anomalous diffraction experiments

Cited by 81 publications

References 16 publications

Crystal Structure and Functional Characterization of Yeast YLR011wp, an Enzyme with NAD(P)H-FMN and Ferric Iron Reductase Activities

Crystal Structure and Functional Characterization of Yeast YLR011wp, an Enzyme with NAD(P)H-FMN and Ferric Iron Reductase Activities

Biochemical and Structural Characterization of the Arabidopsis Bifunctional Enzyme Dethiobiotin Synthetase–Diaminopelargonic Acid Aminotransferase: Evidence for Substrate Channeling in Biotin Synthesis

Genetic and Structural Insights into the Dissemination Potential of the Extremely Broad-Spectrum Class A β-Lactamase KPC-2 Identified in an Escherichia coli Strain and an Enterobacter cloacae Strain Isolated from the Same Patient in France

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