Jose Trincao | Diamond Light Source (original) (raw)
Papers by Jose Trincao
Dalton Transactions, 2008
Two O,S-donor ligands, hydroxythiopyrone and hydroxythiopyridinone derivatives, were developed an... more Two O,S-donor ligands, hydroxythiopyrone and hydroxythiopyridinone derivatives, were developed and studied, as well as the corresponding O,O-derivatives, with a view to their potential pharmacological applications as xanthine oxidase (XO) inhibitors. The biological assays revealed that the O,S-ligands present high inhibitory activity towards XO (nanomolar order, close to that of the pharmaceutical drug allopurinol), in contrast to the corresponding O,O-analogues. Due to the biomedical relevance of this molybdenum-containing enzyme, the corresponding Mo(VI) complexes were studied both in solution and in the solid state, aimed at identifying the source of the biological properties. The solution studies showed that, in comparison with the O,O-analogues, the Mo(VI) complexes with the O,S-ligands present some stabilization, which is even more pronounced for the reduced Mo(IV) species. The crystal structures of the Mo(VI) complexes with the hydroxythiopyrone revealed good flexibility of the coordination modes, with two structural isomers and two polymorphic forms for a mononuclear and a binuclear species, respectively. These results give some support to mechanistic proposals for the XO inhibition involving the interaction of the thione group with the molybdenum cofactor, thus indicating a role of the sulfur atom in the XO inhibition. ‡ Electronic supplementary information (ESI) available: Table S1, containing global formation constants for the hydrolytic species formed in the H + -MoO 4 2− system; showing a cyclic voltammogram of MoO 2 (thiomaltol) 2 ; Tables S3-S8, including more detailed data of compound a-1; illustrating compound a-1; Tables S10-S15 with detailed data of compound b-1; showing compound b-1. See DOI: 10.1039/b717172b dysfunction, hypertension and heart failure; 5 XO catalyses the oxidative hydroxylation of purine substrates, at the molybdenum centre of the pterin cofactor, with the molybdenum coordinated to the cis-dithiolene group of one pyranopterin plus additional oxo, sulfido and hydroxo groups (see Scheme 1). 2,4,6 That oxidative process generates reactive oxygen species (ROS), which can cause cell membrane disintegration, membrane protein damage and DNA mutation, initiating or propagating the development of diseases such as liver injury and cancer. Scheme 1 Schematic representation of the Mo active site and the pyranopterin (molybdopterin) cofactor of xanthine oxidase.
Acta Crystallographica Section A, 2005
Biological Chemistry, 2001
Two ferredoxin genes, fdA and fdB, from the extremely thermoacidophilic crenarchaeon Acidianus am... more Two ferredoxin genes, fdA and fdB, from the extremely thermoacidophilic crenarchaeon Acidianus ambivalens have been sequenced; the sequences share 86% similarity. Whereas the deduced protein sequence of the ferredoxin FdA clearly contains a zinc-binding motif, the corresponding sequence of the FdB is devoid of this motif. Thus far, only the zinc-containing ferredoxin, FdA, from A. ambivalens has been chemically and functionally characterized from its native source. Using RT-PCR and Northern blot analysis, we show that both ferredoxins are expressed by A. ambivalens under either anaerobic or aerobic growth conditions. The zinc-free ferredoxin, FdB, was overexpressed in E. coli and purified to homogeneity. Using EPR spectroscopy, we could demonstrate that FdB contains one [3Fe-4S](1+/0) and one [4Fe-4S](2+/1+) cluster. The reduction potential of the [3Fe-4S](1+/0) cluster was determined as -235+/-10 mV, at pH 6.5, by EPR-monitored redox titration. The high melting temperature of 108+/-2 degrees C of FdB determined by CD spectroscopy reveals that it is not the binding of the Zn2+ that induces the extreme thermostability of these ferredoxins.
Journal of Molecular Biology, 2011
The periplasmic nitrate reductase (NapAB) from Cupriavidus necator is a heterodimeric protein tha... more The periplasmic nitrate reductase (NapAB) from Cupriavidus necator is a heterodimeric protein that belongs to the dimethyl sulfoxide reductase family of mononuclear Mo-containing enzymes and catalyzes the reduction of nitrate to nitrite. The protein comprises a large catalytic subunit (NapA, 91 kDa) containing the molybdenum active site plus one [4Fe-4S] cluster, as well as a small subunit (NapB, 17 kDa), which is a diheme c-type cytochrome involved in electron transfer. Crystals of the oxidized form of the enzyme diffracted beyond 1.5 Å at the European Synchrotron Radiation Facility. This is the highest resolution reported to date for a nitrate reductase, providing true atomic details of the protein active center, and this showed further evidence on the molybdenum coordination sphere, corroborating previous data on the related Desulfovibrio desulfuricans NapA. The molybdenum atom is bound to a total of six sulfur atoms, with no oxygen ligands or water molecules in the vicinity. In the present work, we were also able to prepare partially reduced crystals that revealed two alternate conformations of the Mo-coordinating cysteine. This crystal form was obtained by soaking dithionite into crystals grown in the presence of the ionic liquid [C 4 mim]Cl − . In addition, UV-Vis and EPR spectroscopy studies showed that the periplasmic nitrate reductase from C. necator might work at unexpectedly high redox potentials when compared to all periplasmic nitrate reductases studied to date.
Journal of Biological Inorganic Chemistry, 2006
Superoxide reductase (SOR) is a metalloprotein containing a non-heme iron centre, responsible for... more Superoxide reductase (SOR) is a metalloprotein containing a non-heme iron centre, responsible for the scavenging of superoxide radicals in the cell. The crystal structure of Treponema pallidum (Tp) SOR was determined using soft X-rays and synchrotron radiation. Crystals of the oxidized form were obtained using poly(ethylene glycol) and MgCl2 and diffracted beyond 1.55 Å resolution. The overall architecture is very similar to that of other known SORs but TpSOR contains an N-terminal domain in which the desulforedoxin-type Fe centre, found in other SORs, is absent. This domain conserves the β-barrel topology with an overall arrangement very similar to that of other SOR proteins where the centre is present. The absence of the iron ion and its ligands, however, causes a decrease in the cohesion of the domain and some disorder is observed, particularly in the region where the metal would be harboured. The C-terminal domain exhibits the characteristic immunoglobulin-like fold and harbours the Fe(His)4(Cys) active site. The five ligands of the iron centre are well conserved despite some disorder observed for one of the four molecules in the asymmetric unit. The participation of a glutamate as the sixth ligand of some of the iron centres in Pyrococcus furiosus SOR was not observed in TpSOR. A possible explanation is that either X-ray photoreduction occurred or there was a mixture of redox states at the start of data collection. In agreement with earlier proposals, details in the TpSOR structure also suggest that Lys49 might be involved in attraction of superoxide to the active site.
Acta Crystallographica Section A, 2005
... Page 2. Monte de Caparica, 16/01/2012 Organização: Francisco M. Braz Fernandes, Maria JoãoRom... more ... Page 2. Monte de Caparica, 16/01/2012 Organização: Francisco M. Braz Fernandes, Maria JoãoRomão, Rui MS Martins 2 homo tetramer in the asymmetric unit whereas the half reduced or mixed valance form, Pstmxd, contains only a single molecule in the asymmetric unit. ...
Acta Crystallographica Section F-structural Biology and Crystallization Communications, 2007
The periplasmic nitrate reductase from Cupriavidus necator (also known as Ralstonia eutropha) is ... more The periplasmic nitrate reductase from Cupriavidus necator (also known as Ralstonia eutropha) is a heterodimer that is able to reduce nitrate to nitrite. It comprises a 91 kDa catalytic subunit (NapA) and a 17 kDa subunit (NapB) that is involved in electron transfer. The larger subunit contains a molybdenum active site with a bis-molybdopterin guanine dinucleotide cofactor as well as one [4Fe-4S] cluster, while the small subunit is a di-haem c-type cytochrome. Crystals of the oxidized form of this enzyme were obtained using polyethylene glycol 3350 as precipitant. A single crystal grown at the High Throughput Crystallization Laboratory of the EMBL in Grenoble diffracted to beyond 1.5 Å at the ESRF (ID14-1), which is the highest resolution reported to date for a nitrate reductase. The unit-cell parameters are a = 142.2, b = 82.4, c = 96.8 Å , = 100.7 , space group C2, and one heterodimer is present per asymmetric unit.
Journal of Biological Inorganic Chemistry, 2011
Adenylate kinases (AK) from Gram-negative bacteria are generally devoid of metal ions in their LI... more Adenylate kinases (AK) from Gram-negative bacteria are generally devoid of metal ions in their LID domain. However, three metal ions, zinc, cobalt, and iron, have been found in AK from Gram-negative bacteria. Crystal structures of substrate-free AK from Desulfovibrio gigas with three different metal ions (Zn2+, Zn-AK; Co2+, Co-AK; and Fe2+, Fe-AK) bound in its LID domain have been determined by X-ray crystallography to resolutions 1.8, 2.0, and 3.0 Å, respectively. The zinc and iron forms of the enzyme were crystallized in space group I222, whereas the cobalt-form crystals were C2. The presence of the metals was confirmed by calculation of anomalous difference maps and by X-ray fluorescence scans. The work presented here is the first report of a structure of a metal-containing AK from a Gram-negative bacterium. The native enzyme was crystallized, and only zinc was detected in the LID domain. Co-AK and Fe-AK were obtained by overexpressing the protein in Escherichia coli. Zn-AK and Fe-AK crystallized as monomers in the asymmetric unit, whereas Co-AK crystallized as a dimer. Nevertheless, all three crystal structures are very similar to each other, with the same LID domain topology, the only change being the presence of the different metal atoms. In the absence of any substrate, the LID domain of all holoforms of AK was present in a fully open conformational state. Normal mode analysis was performed to predict fluctuations of the LID domain along the catalytic pathway.
Acta Crystallographica Section F-structural Biology and Crystallization Communications, 2005
Superoxide reductase is a 14 kDa metalloprotein containing a catalytic nonhaem iron centre [Fe(Hi... more Superoxide reductase is a 14 kDa metalloprotein containing a catalytic nonhaem iron centre [Fe(His) 4 Cys]. It is involved in defence mechanisms against oxygen toxicity, scavenging superoxide radicals from the cell. The oxidized form of Treponema pallidum superoxide reductase was crystallized in the presence of polyethylene glycol and magnesium chloride. Two crystal forms were obtained depending on the oxidizing agents used after purification: crystals grown in the presence of K 3 Fe(CN) 6 belonged to space group P2 1 (unit-cell parameters a = 60.3, b = 59.9, c = 64.8 Å , = 106.9 ) and diffracted beyond 1.60 Å resolution, while crystals grown in the presence of Na 2 IrCl 6 belonged to space group C2 (a = 119.4, b = 60.1, c = 65.6 Å , = 104.9 ) and diffracted beyond 1.55 Å . A highly redundant X-ray diffraction data set from the C2 crystal form collected on a copper rotating-anode generator ( = 1.542 Å ) clearly defined the positions of the four Fe atoms present in the asymmetric unit by SAD methods. A MAD experiment at the iron absorption edge confirmed the positions of the previously determined iron sites and provided better phases for model building and refinement. Molecular replacement using the P2 1 data set was successful using a preliminary trace as a search model. A similar arrangement of the four protein molecules could be observed.
Journal of Crystal Growth, 2010
A c c e p t e d m a n u s c r i p t 2 Abstract:
Acta Crystallographica Section F-structural Biology and Crystallization Communications, 2006
In trypanosomatids, trypanothione replaces glutathione in all glutathione-dependent processes. Of... more In trypanosomatids, trypanothione replaces glutathione in all glutathione-dependent processes. Of the two enzymes involved in the glyoxalase pathway, glyoxalase I and glyoxalase II, the latter shows absolute specificity towards trypanothione thioester, making this enzyme an excellent model to understand the molecular basis of trypanothione binding. Cloned glyoxalase II from Leishmania infantum was overexpressed in Escherichia coli, purified and crystallized. Crystals belong to space group C2221 (unit-cell parameters a = 65.6, b = 88.3, c = 85.2 Å) and diffract beyond 2.15 Å using synchrotron radiation. The structure was solved by molecular replacement using the human glyoxalase II structure as a search model. These results, together with future detailed kinetic characterization using lactoyltrypanothione, should shed light on the evolutionary selection of trypanothione instead of glutathione by trypanosomatids.
Dalton Transactions, 2008
Two O,S-donor ligands, hydroxythiopyrone and hydroxythiopyridinone derivatives, were developed an... more Two O,S-donor ligands, hydroxythiopyrone and hydroxythiopyridinone derivatives, were developed and studied, as well as the corresponding O,O-derivatives, with a view to their potential pharmacological applications as xanthine oxidase (XO) inhibitors. The biological assays revealed that the O,S-ligands present high inhibitory activity towards XO (nanomolar order, close to that of the pharmaceutical drug allopurinol), in contrast to the corresponding O,O-analogues. Due to the biomedical relevance of this molybdenum-containing enzyme, the corresponding Mo(VI) complexes were studied both in solution and in the solid state, aimed at identifying the source of the biological properties. The solution studies showed that, in comparison with the O,O-analogues, the Mo(VI) complexes with the O,S-ligands present some stabilization, which is even more pronounced for the reduced Mo(IV) species. The crystal structures of the Mo(VI) complexes with the hydroxythiopyrone revealed good flexibility of the coordination modes, with two structural isomers and two polymorphic forms for a mononuclear and a binuclear species, respectively. These results give some support to mechanistic proposals for the XO inhibition involving the interaction of the thione group with the molybdenum cofactor, thus indicating a role of the sulfur atom in the XO inhibition. ‡ Electronic supplementary information (ESI) available: Table S1, containing global formation constants for the hydrolytic species formed in the H + -MoO 4 2− system; showing a cyclic voltammogram of MoO 2 (thiomaltol) 2 ; Tables S3-S8, including more detailed data of compound a-1; illustrating compound a-1; Tables S10-S15 with detailed data of compound b-1; showing compound b-1. See DOI: 10.1039/b717172b dysfunction, hypertension and heart failure; 5 XO catalyses the oxidative hydroxylation of purine substrates, at the molybdenum centre of the pterin cofactor, with the molybdenum coordinated to the cis-dithiolene group of one pyranopterin plus additional oxo, sulfido and hydroxo groups (see Scheme 1). 2,4,6 That oxidative process generates reactive oxygen species (ROS), which can cause cell membrane disintegration, membrane protein damage and DNA mutation, initiating or propagating the development of diseases such as liver injury and cancer. Scheme 1 Schematic representation of the Mo active site and the pyranopterin (molybdopterin) cofactor of xanthine oxidase.
Acta Crystallographica Section A, 2004
Journal of Inorganic Biochemistry, 2009
Adenylate kinase (AK) mediates the reversible transfer of phosphate groups between the adenylate ... more Adenylate kinase (AK) mediates the reversible transfer of phosphate groups between the adenylate nucleotides and contributes to the maintenance of their constant cellular level, necessary for energy metabolism and nucleic acid synthesis. The AK were purified from crude extracts of two sulfate-reducing bacteria (SRB), Desulfovibrio (D.) gigas NCIB 9332 and Desulfovibrio desulfuricans ATCC 27774, and biochemically and spectroscopically characterised in the native and fully cobalt-or zinc-substituted forms. These are the first reported adenylate kinases that bind either zinc or cobalt and are related to the subgroup of metal-containing AK found, in most cases, in Gram-positive bacteria. The electronic absorption spectrum is consistent with tetrahedral coordinated cobalt, predominantly via sulfur ligands, and is supported by EPR. The involvement of three cysteines in cobalt or zinc coordination was confirmed by chemical methods. Extended X-ray absorption fine structure (EXAFS) indicate that cobalt or zinc are bound by three cysteine residues and one histidine in the metal-binding site of the ''LID" domain. The sequence 129 Cys-X 5 -His-X 15 -Cys-X 2 -Cys of the AK from D. gigas is involved in metal coordination and represents a new type of binding motif that differs from other known zinc-binding sites of AK. Cobalt and zinc play a structural role in stabilizing the LID domain.
Journal of Structural Biology, 2000
Drosophila embryos is controlled, in part, by regulation of translation of mRNAs transcribed in m... more Drosophila embryos is controlled, in part, by regulation of translation of mRNAs transcribed in maternal cells during oogenesis. The Pumilio protein is essential in posterior determination, binding to hunchback mRNA in complex with Nanos to suppress hunchback translation. In order to understand the structural basis of RNA binding, Nanos recruitment, and translational control, we have crystallized a domain of the Drosophila Pumilio protein that binds RNA. The crystals belong to the space group P6 3 with unit cell dimensions of a ؍ b ؍ 94.5 Å, c ؍ 228.9 Å, ␣ ؍  ؍ 90°, ␥ ؍ 120°and diffract to 2.6 Å with synchrotron radiation. We show that the purified protein actively binds RNA and is likely to have a novel RNA binding fold due to a very high content of ␣-helical secondary structure.
Acta Crystallographica Section A, 2005
Key Words lesion bypass, Y-family DNA polymerases, DNA polymerase structures, Rad6-Rad18 enzyme c... more Key Words lesion bypass, Y-family DNA polymerases, DNA polymerase structures, Rad6-Rad18 enzyme complex ■ Abstract This review focuses on eukaryotic translesion synthesis (TLS) DNA polymerases, and the emphasis is on Saccharomyces cerevisiae and human Y-family polymerases (Pols) η, ι, κ, and Rev1, as well as on Polζ , which is a member of the B-family polymerases. The fidelity, mismatch extension ability, and lesion bypass efficiencies of these different polymerases are examined and evaluated in the context of their structures. One major conclusion is that, despite the overall similarity of basic structural features among the Y-family polymerases, there is a high degree of specificity in their lesion bypass properties. Some are able to bypass a particular DNA lesion, whereas others are efficient at only the insertion step or the extension step of lesion bypass. This functional divergence is related to the differences in their structures. Polζ is a highly specialized polymerase specifically adapted for extending primer termini opposite from a diverse array of DNA lesions, and depending upon the DNA lesion, it contributes to lesion bypass in a mutagenic or in an error-free manner. Proliferating cell nuclear antigen (PCNA) provides the central scaffold to which TLS polymerases bind for access to the replication ensemble stalled at a lesion site, and Rad6-Rad18dependent protein ubiquitination is important for polymerase exchange.
Molecular Cell, 2001
The discovery of Pol has gained added significance New York, New York 10029 with the subsequent f... more The discovery of Pol has gained added significance New York, New York 10029 with the subsequent finding that mutations in Pol are 2 Sealy Center for Molecular Science responsible for an inherited disorder, the variant form of University of Texas Medical Branch xeroderma pigmentosum (XP-V; Johnson et al., 1999a; Galveston, Texas 77555 Masutani et al., 1999). Xeroderma pigmentosum (XP) patients are hypersensitive to sunlight, and suffer from a high incidence of skin cancers. In most of these patients Summary (belonging to groups XP-A to XP-G), the disease results from defects in one of the seven genes involved in nucle-DNA polymerase is unique among eukaryotic polyotide excision repair (NER; Freidberg et al., 1995). Howmerases in its proficient ability to replicate through a ever, in %02ف of XP patients, the NER pathway is normal variety of distorting DNA lesions. We report here the but they are defective in their ability to replicate UVcrystal structure of the catalytic core of S. cerevisiae damaged DNA (Lehmann et al., 1975; Cordeiro-Stone DNA polymerase , determined at 2.25Å resolution. et al., 1997). In the majority of cell lines derived from The structure reveals a novel polydactyl right hand-XP-V patients, Pol is severely truncated (Johnson et shaped molecule with a unique polymerase-associal., 1999a; Masutani et al., 1999), resulting in a protein ated domain. We identify the catalytic residues and with no polymerase activity. Pol, thus, is the first DNA show that the fingers and thumb domains are unusupolymerase demonstrated to act as a tumor suppressor ally small and stubby. In particular, the unexpected in humans. absence of helices "O" and "O1" in the fingers domain Yeast and human Pol replicate through a UV-induced suggests that openness of the active site is the critical T-T dimer with the same efficiency and fidelity as on feature which enables DNA polymerase to replicate undamaged DNA. Both polymerases insert A's opposite through DNA lesions such as a UV-induced cis-syn the two T's of the dimer, and on damaged as well as thymine-thymine dimer. undamaged DNA, they incorporate wrong nucleotides with the same frequency of 01ف Ϫ2 -10 Ϫ3 (Washington et Introduction al., 1999, 2000; Johnson et al., 2000c). Yeast Pol can also efficiently and accurately replicate DNA containing The survival of organisms depends critically on the abil-7,8-dihydro-8-oxoguanine (8-oxoG) adducts formed by ity to faithfully replicate DNA. However, cellular DNA is oxidative damage (Haracska et al.,
Biochemical and Biophysical Research Communications, 2002
During Drosophila embryogenesis, Smaug protein represses translation of Nanos through an interact... more During Drosophila embryogenesis, Smaug protein represses translation of Nanos through an interaction with a specific element in its 3 0 UTR. The repression occurs in the bulk cytoplasm of the embryo; Nanos is, however, successfully translated in the specialized cytoplasm of the posterior pole. This generates a gradient of Nanos emanating from the posterior pole that is essential for organizing proper abdominal segmentation. To understand the structural basis of RNA binding and translational control, we have crystallized a domain of Drosophila Smaug that binds RNA. The crystals belong to the space group R3 with unit cell dimensions of
Structure (London, England : 1993), 2004
Cytochrome c peroxidase (CCP) catalyses the reduction of H(2)O(2) to H(2)O, an important step in ... more Cytochrome c peroxidase (CCP) catalyses the reduction of H(2)O(2) to H(2)O, an important step in the cellular detoxification process. The crystal structure of the di-heme CCP from Pseudomonas nautica 617 was obtained in two different conformations in a redox state with the electron transfer heme reduced. Form IN, obtained at pH 4.0, does not contain Ca(2+) and was refined at 2.2 A resolution. This inactive form presents a closed conformation where the peroxidatic heme adopts a six-ligand coordination, hindering the peroxidatic reaction from taking place. Form OUT is Ca(2+) dependent and was crystallized at pH 5.3 and refined at 2.4 A resolution. This active form shows an open conformation, with release of the distal histidine (His71) ligand, providing peroxide access to the active site. This is the first time that the active and inactive states are reported for a di-heme peroxidase.
Acta crystallographica. Section F, Structural biology and crystallization communications, 2008
Dalton Transactions, 2008
Two O,S-donor ligands, hydroxythiopyrone and hydroxythiopyridinone derivatives, were developed an... more Two O,S-donor ligands, hydroxythiopyrone and hydroxythiopyridinone derivatives, were developed and studied, as well as the corresponding O,O-derivatives, with a view to their potential pharmacological applications as xanthine oxidase (XO) inhibitors. The biological assays revealed that the O,S-ligands present high inhibitory activity towards XO (nanomolar order, close to that of the pharmaceutical drug allopurinol), in contrast to the corresponding O,O-analogues. Due to the biomedical relevance of this molybdenum-containing enzyme, the corresponding Mo(VI) complexes were studied both in solution and in the solid state, aimed at identifying the source of the biological properties. The solution studies showed that, in comparison with the O,O-analogues, the Mo(VI) complexes with the O,S-ligands present some stabilization, which is even more pronounced for the reduced Mo(IV) species. The crystal structures of the Mo(VI) complexes with the hydroxythiopyrone revealed good flexibility of the coordination modes, with two structural isomers and two polymorphic forms for a mononuclear and a binuclear species, respectively. These results give some support to mechanistic proposals for the XO inhibition involving the interaction of the thione group with the molybdenum cofactor, thus indicating a role of the sulfur atom in the XO inhibition. ‡ Electronic supplementary information (ESI) available: Table S1, containing global formation constants for the hydrolytic species formed in the H + -MoO 4 2− system; showing a cyclic voltammogram of MoO 2 (thiomaltol) 2 ; Tables S3-S8, including more detailed data of compound a-1; illustrating compound a-1; Tables S10-S15 with detailed data of compound b-1; showing compound b-1. See DOI: 10.1039/b717172b dysfunction, hypertension and heart failure; 5 XO catalyses the oxidative hydroxylation of purine substrates, at the molybdenum centre of the pterin cofactor, with the molybdenum coordinated to the cis-dithiolene group of one pyranopterin plus additional oxo, sulfido and hydroxo groups (see Scheme 1). 2,4,6 That oxidative process generates reactive oxygen species (ROS), which can cause cell membrane disintegration, membrane protein damage and DNA mutation, initiating or propagating the development of diseases such as liver injury and cancer. Scheme 1 Schematic representation of the Mo active site and the pyranopterin (molybdopterin) cofactor of xanthine oxidase.
Acta Crystallographica Section A, 2005
Biological Chemistry, 2001
Two ferredoxin genes, fdA and fdB, from the extremely thermoacidophilic crenarchaeon Acidianus am... more Two ferredoxin genes, fdA and fdB, from the extremely thermoacidophilic crenarchaeon Acidianus ambivalens have been sequenced; the sequences share 86% similarity. Whereas the deduced protein sequence of the ferredoxin FdA clearly contains a zinc-binding motif, the corresponding sequence of the FdB is devoid of this motif. Thus far, only the zinc-containing ferredoxin, FdA, from A. ambivalens has been chemically and functionally characterized from its native source. Using RT-PCR and Northern blot analysis, we show that both ferredoxins are expressed by A. ambivalens under either anaerobic or aerobic growth conditions. The zinc-free ferredoxin, FdB, was overexpressed in E. coli and purified to homogeneity. Using EPR spectroscopy, we could demonstrate that FdB contains one [3Fe-4S](1+/0) and one [4Fe-4S](2+/1+) cluster. The reduction potential of the [3Fe-4S](1+/0) cluster was determined as -235+/-10 mV, at pH 6.5, by EPR-monitored redox titration. The high melting temperature of 108+/-2 degrees C of FdB determined by CD spectroscopy reveals that it is not the binding of the Zn2+ that induces the extreme thermostability of these ferredoxins.
Journal of Molecular Biology, 2011
The periplasmic nitrate reductase (NapAB) from Cupriavidus necator is a heterodimeric protein tha... more The periplasmic nitrate reductase (NapAB) from Cupriavidus necator is a heterodimeric protein that belongs to the dimethyl sulfoxide reductase family of mononuclear Mo-containing enzymes and catalyzes the reduction of nitrate to nitrite. The protein comprises a large catalytic subunit (NapA, 91 kDa) containing the molybdenum active site plus one [4Fe-4S] cluster, as well as a small subunit (NapB, 17 kDa), which is a diheme c-type cytochrome involved in electron transfer. Crystals of the oxidized form of the enzyme diffracted beyond 1.5 Å at the European Synchrotron Radiation Facility. This is the highest resolution reported to date for a nitrate reductase, providing true atomic details of the protein active center, and this showed further evidence on the molybdenum coordination sphere, corroborating previous data on the related Desulfovibrio desulfuricans NapA. The molybdenum atom is bound to a total of six sulfur atoms, with no oxygen ligands or water molecules in the vicinity. In the present work, we were also able to prepare partially reduced crystals that revealed two alternate conformations of the Mo-coordinating cysteine. This crystal form was obtained by soaking dithionite into crystals grown in the presence of the ionic liquid [C 4 mim]Cl − . In addition, UV-Vis and EPR spectroscopy studies showed that the periplasmic nitrate reductase from C. necator might work at unexpectedly high redox potentials when compared to all periplasmic nitrate reductases studied to date.
Journal of Biological Inorganic Chemistry, 2006
Superoxide reductase (SOR) is a metalloprotein containing a non-heme iron centre, responsible for... more Superoxide reductase (SOR) is a metalloprotein containing a non-heme iron centre, responsible for the scavenging of superoxide radicals in the cell. The crystal structure of Treponema pallidum (Tp) SOR was determined using soft X-rays and synchrotron radiation. Crystals of the oxidized form were obtained using poly(ethylene glycol) and MgCl2 and diffracted beyond 1.55 Å resolution. The overall architecture is very similar to that of other known SORs but TpSOR contains an N-terminal domain in which the desulforedoxin-type Fe centre, found in other SORs, is absent. This domain conserves the β-barrel topology with an overall arrangement very similar to that of other SOR proteins where the centre is present. The absence of the iron ion and its ligands, however, causes a decrease in the cohesion of the domain and some disorder is observed, particularly in the region where the metal would be harboured. The C-terminal domain exhibits the characteristic immunoglobulin-like fold and harbours the Fe(His)4(Cys) active site. The five ligands of the iron centre are well conserved despite some disorder observed for one of the four molecules in the asymmetric unit. The participation of a glutamate as the sixth ligand of some of the iron centres in Pyrococcus furiosus SOR was not observed in TpSOR. A possible explanation is that either X-ray photoreduction occurred or there was a mixture of redox states at the start of data collection. In agreement with earlier proposals, details in the TpSOR structure also suggest that Lys49 might be involved in attraction of superoxide to the active site.
Acta Crystallographica Section A, 2005
... Page 2. Monte de Caparica, 16/01/2012 Organização: Francisco M. Braz Fernandes, Maria JoãoRom... more ... Page 2. Monte de Caparica, 16/01/2012 Organização: Francisco M. Braz Fernandes, Maria JoãoRomão, Rui MS Martins 2 homo tetramer in the asymmetric unit whereas the half reduced or mixed valance form, Pstmxd, contains only a single molecule in the asymmetric unit. ...
Acta Crystallographica Section F-structural Biology and Crystallization Communications, 2007
The periplasmic nitrate reductase from Cupriavidus necator (also known as Ralstonia eutropha) is ... more The periplasmic nitrate reductase from Cupriavidus necator (also known as Ralstonia eutropha) is a heterodimer that is able to reduce nitrate to nitrite. It comprises a 91 kDa catalytic subunit (NapA) and a 17 kDa subunit (NapB) that is involved in electron transfer. The larger subunit contains a molybdenum active site with a bis-molybdopterin guanine dinucleotide cofactor as well as one [4Fe-4S] cluster, while the small subunit is a di-haem c-type cytochrome. Crystals of the oxidized form of this enzyme were obtained using polyethylene glycol 3350 as precipitant. A single crystal grown at the High Throughput Crystallization Laboratory of the EMBL in Grenoble diffracted to beyond 1.5 Å at the ESRF (ID14-1), which is the highest resolution reported to date for a nitrate reductase. The unit-cell parameters are a = 142.2, b = 82.4, c = 96.8 Å , = 100.7 , space group C2, and one heterodimer is present per asymmetric unit.
Journal of Biological Inorganic Chemistry, 2011
Adenylate kinases (AK) from Gram-negative bacteria are generally devoid of metal ions in their LI... more Adenylate kinases (AK) from Gram-negative bacteria are generally devoid of metal ions in their LID domain. However, three metal ions, zinc, cobalt, and iron, have been found in AK from Gram-negative bacteria. Crystal structures of substrate-free AK from Desulfovibrio gigas with three different metal ions (Zn2+, Zn-AK; Co2+, Co-AK; and Fe2+, Fe-AK) bound in its LID domain have been determined by X-ray crystallography to resolutions 1.8, 2.0, and 3.0 Å, respectively. The zinc and iron forms of the enzyme were crystallized in space group I222, whereas the cobalt-form crystals were C2. The presence of the metals was confirmed by calculation of anomalous difference maps and by X-ray fluorescence scans. The work presented here is the first report of a structure of a metal-containing AK from a Gram-negative bacterium. The native enzyme was crystallized, and only zinc was detected in the LID domain. Co-AK and Fe-AK were obtained by overexpressing the protein in Escherichia coli. Zn-AK and Fe-AK crystallized as monomers in the asymmetric unit, whereas Co-AK crystallized as a dimer. Nevertheless, all three crystal structures are very similar to each other, with the same LID domain topology, the only change being the presence of the different metal atoms. In the absence of any substrate, the LID domain of all holoforms of AK was present in a fully open conformational state. Normal mode analysis was performed to predict fluctuations of the LID domain along the catalytic pathway.
Acta Crystallographica Section F-structural Biology and Crystallization Communications, 2005
Superoxide reductase is a 14 kDa metalloprotein containing a catalytic nonhaem iron centre [Fe(Hi... more Superoxide reductase is a 14 kDa metalloprotein containing a catalytic nonhaem iron centre [Fe(His) 4 Cys]. It is involved in defence mechanisms against oxygen toxicity, scavenging superoxide radicals from the cell. The oxidized form of Treponema pallidum superoxide reductase was crystallized in the presence of polyethylene glycol and magnesium chloride. Two crystal forms were obtained depending on the oxidizing agents used after purification: crystals grown in the presence of K 3 Fe(CN) 6 belonged to space group P2 1 (unit-cell parameters a = 60.3, b = 59.9, c = 64.8 Å , = 106.9 ) and diffracted beyond 1.60 Å resolution, while crystals grown in the presence of Na 2 IrCl 6 belonged to space group C2 (a = 119.4, b = 60.1, c = 65.6 Å , = 104.9 ) and diffracted beyond 1.55 Å . A highly redundant X-ray diffraction data set from the C2 crystal form collected on a copper rotating-anode generator ( = 1.542 Å ) clearly defined the positions of the four Fe atoms present in the asymmetric unit by SAD methods. A MAD experiment at the iron absorption edge confirmed the positions of the previously determined iron sites and provided better phases for model building and refinement. Molecular replacement using the P2 1 data set was successful using a preliminary trace as a search model. A similar arrangement of the four protein molecules could be observed.
Journal of Crystal Growth, 2010
A c c e p t e d m a n u s c r i p t 2 Abstract:
Acta Crystallographica Section F-structural Biology and Crystallization Communications, 2006
In trypanosomatids, trypanothione replaces glutathione in all glutathione-dependent processes. Of... more In trypanosomatids, trypanothione replaces glutathione in all glutathione-dependent processes. Of the two enzymes involved in the glyoxalase pathway, glyoxalase I and glyoxalase II, the latter shows absolute specificity towards trypanothione thioester, making this enzyme an excellent model to understand the molecular basis of trypanothione binding. Cloned glyoxalase II from Leishmania infantum was overexpressed in Escherichia coli, purified and crystallized. Crystals belong to space group C2221 (unit-cell parameters a = 65.6, b = 88.3, c = 85.2 Å) and diffract beyond 2.15 Å using synchrotron radiation. The structure was solved by molecular replacement using the human glyoxalase II structure as a search model. These results, together with future detailed kinetic characterization using lactoyltrypanothione, should shed light on the evolutionary selection of trypanothione instead of glutathione by trypanosomatids.
Dalton Transactions, 2008
Two O,S-donor ligands, hydroxythiopyrone and hydroxythiopyridinone derivatives, were developed an... more Two O,S-donor ligands, hydroxythiopyrone and hydroxythiopyridinone derivatives, were developed and studied, as well as the corresponding O,O-derivatives, with a view to their potential pharmacological applications as xanthine oxidase (XO) inhibitors. The biological assays revealed that the O,S-ligands present high inhibitory activity towards XO (nanomolar order, close to that of the pharmaceutical drug allopurinol), in contrast to the corresponding O,O-analogues. Due to the biomedical relevance of this molybdenum-containing enzyme, the corresponding Mo(VI) complexes were studied both in solution and in the solid state, aimed at identifying the source of the biological properties. The solution studies showed that, in comparison with the O,O-analogues, the Mo(VI) complexes with the O,S-ligands present some stabilization, which is even more pronounced for the reduced Mo(IV) species. The crystal structures of the Mo(VI) complexes with the hydroxythiopyrone revealed good flexibility of the coordination modes, with two structural isomers and two polymorphic forms for a mononuclear and a binuclear species, respectively. These results give some support to mechanistic proposals for the XO inhibition involving the interaction of the thione group with the molybdenum cofactor, thus indicating a role of the sulfur atom in the XO inhibition. ‡ Electronic supplementary information (ESI) available: Table S1, containing global formation constants for the hydrolytic species formed in the H + -MoO 4 2− system; showing a cyclic voltammogram of MoO 2 (thiomaltol) 2 ; Tables S3-S8, including more detailed data of compound a-1; illustrating compound a-1; Tables S10-S15 with detailed data of compound b-1; showing compound b-1. See DOI: 10.1039/b717172b dysfunction, hypertension and heart failure; 5 XO catalyses the oxidative hydroxylation of purine substrates, at the molybdenum centre of the pterin cofactor, with the molybdenum coordinated to the cis-dithiolene group of one pyranopterin plus additional oxo, sulfido and hydroxo groups (see Scheme 1). 2,4,6 That oxidative process generates reactive oxygen species (ROS), which can cause cell membrane disintegration, membrane protein damage and DNA mutation, initiating or propagating the development of diseases such as liver injury and cancer. Scheme 1 Schematic representation of the Mo active site and the pyranopterin (molybdopterin) cofactor of xanthine oxidase.
Acta Crystallographica Section A, 2004
Journal of Inorganic Biochemistry, 2009
Adenylate kinase (AK) mediates the reversible transfer of phosphate groups between the adenylate ... more Adenylate kinase (AK) mediates the reversible transfer of phosphate groups between the adenylate nucleotides and contributes to the maintenance of their constant cellular level, necessary for energy metabolism and nucleic acid synthesis. The AK were purified from crude extracts of two sulfate-reducing bacteria (SRB), Desulfovibrio (D.) gigas NCIB 9332 and Desulfovibrio desulfuricans ATCC 27774, and biochemically and spectroscopically characterised in the native and fully cobalt-or zinc-substituted forms. These are the first reported adenylate kinases that bind either zinc or cobalt and are related to the subgroup of metal-containing AK found, in most cases, in Gram-positive bacteria. The electronic absorption spectrum is consistent with tetrahedral coordinated cobalt, predominantly via sulfur ligands, and is supported by EPR. The involvement of three cysteines in cobalt or zinc coordination was confirmed by chemical methods. Extended X-ray absorption fine structure (EXAFS) indicate that cobalt or zinc are bound by three cysteine residues and one histidine in the metal-binding site of the ''LID" domain. The sequence 129 Cys-X 5 -His-X 15 -Cys-X 2 -Cys of the AK from D. gigas is involved in metal coordination and represents a new type of binding motif that differs from other known zinc-binding sites of AK. Cobalt and zinc play a structural role in stabilizing the LID domain.
Journal of Structural Biology, 2000
Drosophila embryos is controlled, in part, by regulation of translation of mRNAs transcribed in m... more Drosophila embryos is controlled, in part, by regulation of translation of mRNAs transcribed in maternal cells during oogenesis. The Pumilio protein is essential in posterior determination, binding to hunchback mRNA in complex with Nanos to suppress hunchback translation. In order to understand the structural basis of RNA binding, Nanos recruitment, and translational control, we have crystallized a domain of the Drosophila Pumilio protein that binds RNA. The crystals belong to the space group P6 3 with unit cell dimensions of a ؍ b ؍ 94.5 Å, c ؍ 228.9 Å, ␣ ؍  ؍ 90°, ␥ ؍ 120°and diffract to 2.6 Å with synchrotron radiation. We show that the purified protein actively binds RNA and is likely to have a novel RNA binding fold due to a very high content of ␣-helical secondary structure.
Acta Crystallographica Section A, 2005
Key Words lesion bypass, Y-family DNA polymerases, DNA polymerase structures, Rad6-Rad18 enzyme c... more Key Words lesion bypass, Y-family DNA polymerases, DNA polymerase structures, Rad6-Rad18 enzyme complex ■ Abstract This review focuses on eukaryotic translesion synthesis (TLS) DNA polymerases, and the emphasis is on Saccharomyces cerevisiae and human Y-family polymerases (Pols) η, ι, κ, and Rev1, as well as on Polζ , which is a member of the B-family polymerases. The fidelity, mismatch extension ability, and lesion bypass efficiencies of these different polymerases are examined and evaluated in the context of their structures. One major conclusion is that, despite the overall similarity of basic structural features among the Y-family polymerases, there is a high degree of specificity in their lesion bypass properties. Some are able to bypass a particular DNA lesion, whereas others are efficient at only the insertion step or the extension step of lesion bypass. This functional divergence is related to the differences in their structures. Polζ is a highly specialized polymerase specifically adapted for extending primer termini opposite from a diverse array of DNA lesions, and depending upon the DNA lesion, it contributes to lesion bypass in a mutagenic or in an error-free manner. Proliferating cell nuclear antigen (PCNA) provides the central scaffold to which TLS polymerases bind for access to the replication ensemble stalled at a lesion site, and Rad6-Rad18dependent protein ubiquitination is important for polymerase exchange.
Molecular Cell, 2001
The discovery of Pol has gained added significance New York, New York 10029 with the subsequent f... more The discovery of Pol has gained added significance New York, New York 10029 with the subsequent finding that mutations in Pol are 2 Sealy Center for Molecular Science responsible for an inherited disorder, the variant form of University of Texas Medical Branch xeroderma pigmentosum (XP-V; Johnson et al., 1999a; Galveston, Texas 77555 Masutani et al., 1999). Xeroderma pigmentosum (XP) patients are hypersensitive to sunlight, and suffer from a high incidence of skin cancers. In most of these patients Summary (belonging to groups XP-A to XP-G), the disease results from defects in one of the seven genes involved in nucle-DNA polymerase is unique among eukaryotic polyotide excision repair (NER; Freidberg et al., 1995). Howmerases in its proficient ability to replicate through a ever, in %02ف of XP patients, the NER pathway is normal variety of distorting DNA lesions. We report here the but they are defective in their ability to replicate UVcrystal structure of the catalytic core of S. cerevisiae damaged DNA (Lehmann et al., 1975; Cordeiro-Stone DNA polymerase , determined at 2.25Å resolution. et al., 1997). In the majority of cell lines derived from The structure reveals a novel polydactyl right hand-XP-V patients, Pol is severely truncated (Johnson et shaped molecule with a unique polymerase-associal., 1999a; Masutani et al., 1999), resulting in a protein ated domain. We identify the catalytic residues and with no polymerase activity. Pol, thus, is the first DNA show that the fingers and thumb domains are unusupolymerase demonstrated to act as a tumor suppressor ally small and stubby. In particular, the unexpected in humans. absence of helices "O" and "O1" in the fingers domain Yeast and human Pol replicate through a UV-induced suggests that openness of the active site is the critical T-T dimer with the same efficiency and fidelity as on feature which enables DNA polymerase to replicate undamaged DNA. Both polymerases insert A's opposite through DNA lesions such as a UV-induced cis-syn the two T's of the dimer, and on damaged as well as thymine-thymine dimer. undamaged DNA, they incorporate wrong nucleotides with the same frequency of 01ف Ϫ2 -10 Ϫ3 (Washington et Introduction al., 1999, 2000; Johnson et al., 2000c). Yeast Pol can also efficiently and accurately replicate DNA containing The survival of organisms depends critically on the abil-7,8-dihydro-8-oxoguanine (8-oxoG) adducts formed by ity to faithfully replicate DNA. However, cellular DNA is oxidative damage (Haracska et al.,
Biochemical and Biophysical Research Communications, 2002
During Drosophila embryogenesis, Smaug protein represses translation of Nanos through an interact... more During Drosophila embryogenesis, Smaug protein represses translation of Nanos through an interaction with a specific element in its 3 0 UTR. The repression occurs in the bulk cytoplasm of the embryo; Nanos is, however, successfully translated in the specialized cytoplasm of the posterior pole. This generates a gradient of Nanos emanating from the posterior pole that is essential for organizing proper abdominal segmentation. To understand the structural basis of RNA binding and translational control, we have crystallized a domain of Drosophila Smaug that binds RNA. The crystals belong to the space group R3 with unit cell dimensions of
Structure (London, England : 1993), 2004
Cytochrome c peroxidase (CCP) catalyses the reduction of H(2)O(2) to H(2)O, an important step in ... more Cytochrome c peroxidase (CCP) catalyses the reduction of H(2)O(2) to H(2)O, an important step in the cellular detoxification process. The crystal structure of the di-heme CCP from Pseudomonas nautica 617 was obtained in two different conformations in a redox state with the electron transfer heme reduced. Form IN, obtained at pH 4.0, does not contain Ca(2+) and was refined at 2.2 A resolution. This inactive form presents a closed conformation where the peroxidatic heme adopts a six-ligand coordination, hindering the peroxidatic reaction from taking place. Form OUT is Ca(2+) dependent and was crystallized at pH 5.3 and refined at 2.4 A resolution. This active form shows an open conformation, with release of the distal histidine (His71) ligand, providing peroxide access to the active site. This is the first time that the active and inactive states are reported for a di-heme peroxidase.
Acta crystallographica. Section F, Structural biology and crystallization communications, 2008