Alves, T, Besson S, Duarte LC, Pettigrew GW, Girio FMF, Devreese B, Vandenberghe I, Van Beeumen J, Fauque G, Moura I.
1999.
A cytochrome c peroxidase from Pseudomonas nautica 617 active at high ionic strength: expression, purification and characterization, Oct 12. Biochimica Et Biophysica Acta-Protein Structure and Molecular Enzymology. 1434:248-259., Number 2
AbstractCytochrome c peroxidase was expressed in cells of Pseudomonas nautica strain 617 grown under microaerophilic conditions. The 36.5 kDa dihaemic enzyme was purified to electrophoretic homogeneity in three chromatographic steps. N-terminal sequence comparison showed that the Ps. nautica enzyme exhibits a high similarity with the corresponding proteins from Paracoccus denitrificans and Pseudomonas aeruginosa. UV-visible spectra confirm calcium activation of the enzyme through spin state transition of the peroxidatic haem. Monohaemic cytochrome c(552) from Ps. nautica was identified as the physiological electron donor, with a half-saturating concentration of 122 mu M and allowing a maximal catalytic centre activity of 116 000 min(-1). Using this cytochrome the enzyme retained the same activity even at high ionic strength. There are indications that the interactions between the two redox partners are mainly hydrophobic in nature. (C) 1999 Elsevier Science B.V. All rights reserved.
Dias, JM, Than ME, Humm A, Huber R, Bourenkov GP, Bartunik HD, Bursakov S, Calvete J, Caldeira J, Carneiro C, Moura JJ, Moura I, Romao MJ.
1999.
Crystal structure of the first dissimilatory nitrate reductase at 1.9 A solved by MAD methods, Jan 15. Structure. 7:65-79., Number 1
AbstractBACKGROUND: The periplasmic nitrate reductase (NAP) from the sulphate reducing bacterium Desulfovibrio desulfuricans ATCC 27774 is induced by growth on nitrate and catalyses the reduction of nitrate to nitrite for respiration. NAP is a molybdenum-containing enzyme with one bis-molybdopterin guanine dinucleotide (MGD) cofactor and one [4Fe-4S] cluster in a single polypeptide chain of 723 amino acid residues. To date, there is no crystal structure of a nitrate reductase. RESULTS: The first crystal structure of a dissimilatory (respiratory) nitrate reductase was determined at 1.9 A resolution by multiwavelength anomalous diffraction (MAD) methods. The structure is folded into four domains with an alpha/beta-type topology and all four domains are involved in cofactor binding. The [4Fe-4S] centre is located near the periphery of the molecule, whereas the MGD cofactor extends across the interior of the molecule interacting with residues from all four domains. The molybdenum atom is located at the bottom of a 15 A deep crevice, and is positioned 12 A from the [4Fe-4S] cluster. The structure of NAP reveals the details of the catalytic molybdenum site, which is coordinated to two MGD cofactors, Cys140, and a water/hydroxo ligand. A facile electron-transfer pathway through bonds connects the molybdenum and the [4Fe-4S] cluster. CONCLUSIONS: The polypeptide fold of NAP and the arrangement of the cofactors is related to that of Escherichia coli formate dehydrogenase (FDH) and distantly resembles dimethylsulphoxide reductase. The close structural homology of NAP and FDH shows how small changes in the vicinity of the molybdenum catalytic site are sufficient for the substrate specificity.
Almendra, MJ, Brondino CD, Gavel O, Pereira AS, Tavares P, Bursakov S, Duarte R, Caldeira J, Moura JJ, Moura I.
1999.
Purification and characterization of a tungsten-containing formate dehydrogenase from Desulfovibrio gigas, Dec 7. Biochemistry. 38:16366-72., Number 49
AbstractAn air-stable formate dehydrogenase (FDH), an enzyme that catalyzes the oxidation of formate to carbon dioxide, was purified from the sulfate reducing organism Desulfovibrio gigas (D. gigas) NCIB 9332. D. gigas FDH is a heterodimeric protein [alpha (92 kDa) and beta (29 kDa) subunits] and contains 7 +/- 1 Fe/protein and 0.9 +/- 0.1 W/protein. Selenium was not detected. The UV/visible absorption spectrum of D. gigas FDH is typical of an iron-sulfur protein. Analysis of pterin nucleotides yielded a content of 1.3 +/- 0.1 guanine monophosphate/mol of enzyme, which suggests a tungsten coordination with two molybdopterin guanine dinucleotide cofactors. Both Mossbauer spectroscopy performed on D. gigas FDH grown in a medium enriched with (57)Fe and EPR studies performed in the native and fully reduced state of the protein confirmed the presence of two [4Fe-4S] clusters. Variable-temperature EPR studies showed the presence of two signals compatible with an atom in a d(1) configuration albeit with an unusual relaxation behavior as compared to the one generally observed for W(V) ions.
Dias, JM, Bursakov S, Carneiro C, Moura JJ, Moura I, Romao MJ.
1999.
Crystallization and preliminary x-ray analysis of a nitrate reductase from Desulfovibrio desulfuricans ATCC 27774, Apr. Acta Crystallogr D Biol Crystallogr. 55:877-9., Number Pt 4
AbstractPeriplasmic nitrate reductase from the sulfate-reducing bacterium Desulfovibrio desulfuricans ATCC 27774 contains two molybdopterin guanine dinucleotide cofactors and one [4Fe-4S] cluster as prosthetic groups and catalyzes the conversion of nitrate to nitrite. Crystals of the oxidized form of this enzyme were obtained using PEG as precipitant and belong to space group P3121 or P3221, with unit-cell dimensions a = b = 106.3, c = 135.1 A. There is one monomer of 80 kDa in the asymmetric unit, which corresponds to a Matthews ratio of 2.75 A3 Da-1. Using cryo-cooling procedures and X-rays from a rotating-anode generator, diffraction was observed to beyond 3.0 A resolution.
Bursakov, SA, Brondino C, Dias JM, Carneiro C, Caldeira J, Duarte RO, Romao MJ, Moura I, Moura JJG.
1999.
Cross immunological reactions and spectroscopy study within nitrate reductase and other mononuclear Mo containing enzymes of the sulfate reducing bacteria. Journal of Inorganic Biochemistry. 74:86-86., Number 1-4
Abstractn/a
Dias, JM, Than ME, Humm A, Huber R, Bourenkov GP, Bartunik HD, Bursakov S, Calvete J, Caldeira J, Carneiro C, Moura JJG, Moura I, Romao MJ.
1999.
Crystal structure of the first dissimilatory nitrate reductase at 1.9 angstrom solved by MAD methods. Structure with Folding & Design. 7:65-79., Number 1
Abstractn/a
Dias, JM, Than ME, Huber R, Bourenkov GP, Bartunik HD, Bursakov S, Moura JJG, Moura I, Romao MJ.
1999.
Crystallographic studies of a dissimilatory nitrate reductase and mechanistic implications. Journal of Inorganic Biochemistry. 74:113-113., Number 1-4
Abstractn/a
Teixeira, S, Dias JM, Carvalho AL, Bourenkov G, Bartunik H, Almendra MJ, Moura I, Moura JJG, Romao MJ.
1999.
Crystallographic studies on a tungsten-containning formate dehydrogenase from Desulfovibrio gigas. Journal of Inorganic Biochemistry. 74:89-89., Number 1-4
Abstractn/a
Romao, MJ, Carvalho AL, Dias JM, Teixeira S, Bourenkov G, Bartunik H, Huber R, Maia L, Mira L.
1999.
Preliminary crystallographic studies of xanthine oxidase purified from rat liver. Journal of Inorganic Biochemistry. 74:281-281., Number 1-4
Abstractn/a
Almendra, MJ, Brondino CD, Gavel O, Pereira AS, Tavares P, Bursakov S, Duarte R, Caldeira J, Moura JJG, Moura I.
1999.
Purification and characterization of a tungsten-containing formate dehydrogenase from Desulfovibrio gigas. Biochemistry. {38}:{16366-16372}., Number {49}
AbstractAn air-stable formate dehydrogenase (FDH), an enzyme that catalyzes the oxidation of formate to carbon dioxide, was purified from the sulfate reducing organism Desulfovibrio gigas (D. gigas) NCIB 9332. D. gigas FDH is a heterodimeric protein [alpha (92 kDa) and beta (29 kDa) subunits] and contains 7 +/- 1 Fe/protein and 0.9 +/- 0.1 W/protein, Selenium was not detected. The UV/visible absorption spectrum of D, gigas FDH is typical of an iron-sulfur protein. Analysis of pterin nucleotides yielded a content of 1.3 +/- 0.1 guanine monophosphate/mol of enzyme, which suggests a tungsten coordination with two molybdopterin guanine dinucleotide cofactors. Both Mossbauer spectroscopy performed on D. gigas FDH grown in a medium enriched with Fe-57 and EPR studies performed in the native and fully reduced state of the protein confirmed the presence of two [4Fe-4S] clusters. Variable-temperature EPR studies showed the presence of two signals compatible with an atom in a d(1) configuration albeit with an unusual relaxation behavior as compared to the one generally observed for W(V) ions.