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M J Romão

Publications and source records attributed to M J Romão.

At least 19 recordsLinked to original sources
PubMed2001

Purification, crystallization and identification by X-ray analysis of a prostate kallikrein from horse seminal plasma.

The purification, crystallization and identification by X-ray diffraction analysis of a horse kallikrein is reported. The protein was purified from horse seminal plasma. Crystals belong to space group C2 and the structure was solved by the MIRAS method, with two heavy-atom derivatives of mercury and platinum. X-ray diffraction data to 1.42 A resolution were collected at the ESRF synchrotron-radiation source.

Amino Acid Sequence↗
PubMed2001

Tungsten-containing formate dehydrogenase from Desulfovibrio gigas: metal identification and preliminary structural data by multi-wavelength crystallography.

The tungsten-containing formate dehydrogenase (W-FDH) isolated from Desulfovibrio gigas has been crystallized in space group P2(1), with cell parameters a = 73.8 A, b = 111.3 A, c = 156.6 A and beta = 93.7 degrees. These crystals diffract to beyond 2.0 A on a synchrotron radiation source. W-FDH is a heterodimer (92 kDa and 29 kDa subunits) and two W-FDH molecules are present in the asymmetric unit. Although a molecular replacement solution was found using the periplasmic nitrate reductase as a search model, additional phasing information was needed. A multiple-wavelength anomalous dispersion (MAD) dataset was collected at the W- and Fe-edges, at four different wavelengths. Anomalous and dispersive difference data allowed us to unambiguously identify the metal atoms bound to W-FDH as one W atom with a Se-cysteine ligand as well as one [4Fe-4S] cluster in the 92 kDa subunit, and three additional [4Fe-4S] centers in the smaller 29 kDa subunit. The D. gigas W-FDH was previously characterized based on metal analysis and spectroscopic data. One W atom was predicted to be bound to two molybdopterin guanine dinucleotide (MGD) pterin cofactors and two [4Fe-4S] centers were proposed to be present. The crystallographic data now reported reveal a selenium atom (as a Se-cysteine) coordinating to the W site, as well as two extra [4Fe-4S] clusters not anticipated before. The EPR data were re-evaluated in the light of these new results.

Crystallography, X-Ray↗
PubMed2001

Structure refinement of the aldehyde oxidoreductase from Desulfovibrio gigas (MOP) at 1.28 A.

The sulfate-reducing bacterium aldehyde oxidoreductase from Desulfovibrio gigas (MOP) is a member of the xanthine oxidase family of enzymes. It has 907 residues on a single polypeptide chain, a molybdopterin cytosine dinucleotide (MCD) cofactor and two [2Fe-2S] iron-sulfur clusters. Synchrotron data to almost atomic resolution were collected for improved cryo-cooled crystals of this enzyme in the oxidized form. The cell constants of a=b=141.78 A and c=160.87 A are about 2% shorter than those of room temperature data, yielding 233,755 unique reflections in space group P6(1)22, at 1.28 A resolution. Throughout the entire refinement the full gradient least-squares method was used, leading to a final R factor of 14.5 and Rfree factor of 19.3 (4sigma cut-off) with "riding" H-atoms at their calculated positions. The model contains 8146 non-hydrogen atoms described by anisotropic displacement parameters with an observations/parameters ratio of 4.4. It includes alternate conformations for 17 amino acid residues. At 1.28 A resolution, three Cl- and two Mg2+ ions from the crystallization solution were clearly identified. With the exception of one Cl- which is buried and 8 A distant from the Mo atom, the other ions are close to the molecular surface and may contribute to crystal packing. The overall structure has not changed in comparison to the lower resolution model apart from local corrections that included some loop adjustments and alternate side-chain conformations. Based on the estimated errors of bond distances obtained by blocked least-squares matrix inversion, a more detailed analysis of the three redox centres was possible. For the MCD cofactor, the resulting geometric parameters confirmed its reduction state as a tetrahydropterin. At the Mo centre, estimated corrections calculated for the Fourier ripples artefact are very small when compared to the experimental associated errors, supporting the suggestion that the fifth ligand is a water molecule rather than a hydroxide. Concerning the two iron-sulfur centres, asymmetry in the Fe-S distances as well as differences in the pattern of NH.S hydrogen-bonding interactions was observed, which influences the electron distribution upon reduction and causes non-equivalence of the individual Fe atoms in each cluster.

Aldehyde Oxidoreductases↗
PubMed2000

Gene sequence and crystal structure of the aldehyde oxidoreductase from Desulfovibrio desulfuricans ATCC 27774.

The aldehyde oxidoreductase (MOD) isolated from the sulfate reducer Desulfovibrio desulfuricans (ATCC 27774) is a member of the xanthine oxidase family of molybdenum-containing enzymes. It has substrate specificity similar to that of the homologous enzyme from Desulfovibrio gigas (MOP) and the primary sequences from both enzymes show 68 % identity. The enzyme was crystallized in space group P6(1)22, with unit cell dimensions of a=b=156.4 A and c=177.1 A, and diffraction data were obtained to beyond 2.8 A. The crystal structure was solved by Patterson search techniques using the coordinates of the D. gigas enzyme. The overall fold of the D. desulfuricans enzyme is very similar to MOP and the few differences are mapped to exposed regions of the molecule. This is reflected in the electrostatic potential surfaces of both homologous enzymes, one exception being the surface potential in a region identifiable as the putative docking site of the physiological electron acceptor. Other essential features of the MOP structure, such as residues of the active-site cavity, are basically conserved in MOD. Two mutations are located in the pocket bearing a chain of catalytically relevant water molecules. As deduced from this work, both these enzymes are very closely related in terms of their sequences as well as 3D structures. The comparison allowed confirmation and establishment of features that are essential for their function; namely, conserved residues in the active-site, catalytically relevant water molecules and recognition of the physiological electron acceptor docking site.

Aldehyde Oxidoreductases↗
PubMed2000

Biochemical/spectroscopic characterization and preliminary X-ray analysis of a new aldehyde oxidoreductase isolated from Desulfovibrio desulfuricans ATCC 27774.

Aldehyde oxidoreductase (AOR) activity has been found in different sulfate reducing organisms (Moura, J. J. G., and Barata, B. A. S. (1994) in Methods in Enzymology (Peck, H. D., Jr., and LeGall, J., Eds.), Vol. 243, Chap. 4. Academic Press; Romão, M. J., Knäblein, J., Huber, R., and Moura, J. J. G. (1997) Prog. Biophys. Mol. Biol. 68, 121-144). The enzyme was purified to homogeneity from extracts of Desulfovibrio desulfuricans (Dd) ATCC 27774, a sulfate reducer that can use sulfate or nitrate as terminal respiratory substrates. The protein (AORDd) is described as a homodimer (monomer, circa 100 kDa), contains a Mo-MCD pterin, 2 x [2Fe-2S] clusters, and lacks a flavin group. Visible and EPR spectroscopies indicate a close similarity with the AOR purified from Desulfovibrio gigas (Dg) (Barata, B. A. S., LeGall, J., and Moura, J. J. G. (1993) Biochemistry 32, 11559-11568). Activity and substrate specificity for different aldehydes were determined. EPR studies were performed in native and reduced states of the enzyme and after treatment with ethylene glycol and dithiothreitol. The AORDd was crystallized using ammonium sulfate as precipitant and the crystals belong to the space group P6(1)22, with unit cell dimensions a = b = 156.4 and c = 177.1 A. These crystals diffract to beyond 2.5 A resolution and a full data set was measured on a rotating anode generator. The data were used to solve the structure by Patterson Search methods, using the model of AORDg.

Aldehyde Oxidoreductases↗
PubMed2000

Ionic strength dependence of the non-physiological electron transfer between flavodoxin and cytochrome c553 from D vulgaris.

A hypothetical model for the non-physiological electron transfer complex between cytochrome c553 (c553) and the flavodoxin (fld) from the sulphate-reducing bacteria Desulfovibrio vulgaris has been recently published [1] based on rigid-body docking and refined by molecular dynamics. In this study, the functional validity of this model is tested by looking at the role of electrostatics in the non-physiological interprotein electron transfer between the two proteins at different ionic strengths. The results are compared with the electron transfer between fld and cytochrome c from horse heart (hhc). Second-order rate constants (k2) were measured for both non-physiological systems at different ionic strengths: a complex, bell-shaped behaviour is observed for the k2 of the c553/fld redox pair with an optimum rate at I=58 mmol l(-1), whereas under the same conditions the k2 for hhc/fld decreased monotonically with increasing ionic strength. Results from the electron transfer kinetics are rationalised in terms of reorganisational effects of an ensemble of conformations of the electron transfer competent c553/fld complexes, consistent with the published model.

Cytochrome c Group↗
PubMed2000

Crystallization and preliminary X-ray analysis of a membrane-bound nitrite reductase from Desulfovibrio desulfuricans ATCC 27774.

Nitrite reductase from the sulfate-reducing bacterium Desulfovibrio desulfuricans ATCC 27774 is a multihaem (type c) membrane-bound enzyme that catalyzes the dissimilatory conversion of nitrite to ammonia. Crystals of the oxidized form of this enzyme were obtained using PEG and CaCl(2) as precipitants in the presence of 3--(decylmethylammonium)propane-1-sulfonate and belong to the space group P2(1)2(1)2(1), with unit-cell parameters a = 78.94, b = 104.59, c = 143.18 A. A complete data set to 2.30 A resolution was collected using synchrotron radiation at the ESRF. However, the crystals may diffract to beyond 1.7 A and high-resolution data will be collected in the near future.

Bacterial Proteins↗
PubMed1999

Crystal structure of the first dissimilatory nitrate reductase at 1.9 A solved by MAD methods.

BACKGROUND: 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.

Amino Acid Sequence↗
PubMed1999

Effects of protein-protein interactions on electron transfer: docking and electron transfer calculations for complexes between flavodoxin and c-type cytochromes.

Theoretical studies of protein-protein association and electron transfer were performed on the binary systems formed by Desulfovibrio vulgaris Hildenborough (D. v. H.) flavodoxin and D. v. H. cytochrome c553 and by flavodoxin and horse heart cytochrome c. Initial structures for the complexes were obtained by rigid-body docking and were refined by MD to allow for molecular flexibility. The structures thus obtained were analysed in terms of their relative stability through the calculation of excess energies. Electrostatic, van der Waals and solvation energy terms showed all to have significant contributions to the stability of complexes. In the best association solutions found for both cytochromes, these bind to different zones of flavodoxin. The binding site of flavodoxin observed for cytochrome c is in accordance with earlier works [27]. The various association modes found were characterised in terms of electron transfer using the Pathways model. For complexes between flavodoxin and horse heart cytochrome c, some correlation was observed between electron tunnelling coupling factors and conformation energy; the best conformation found for electron transfer corresponded also to the best one in terms of energy. For complexes between flavodoxin and cytochrome c553 this was not the case and a lower correlation was observed between electron tunnelling coupling factors and excess energies. These results are in accordance with the differences in the experimental dependence of electron transfer rates with ionic strength observed between these two cases.

Cytochrome c Group↗
PubMed1999

Crystallization and preliminary x-ray analysis of a nitrate reductase from Desulfovibrio desulfuricans ATCC 27774.

Periplasmic 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.

Bacterial Proteins↗
PubMed1999

Structural studies by X-ray diffraction on metal substituted desulforedoxin, a rubredoxin-type protein.

Desulforedoxin (Dx), isolated from the sulfate reducing bacterium Desulfovibrio gigas, is a small homodimeric (2 x 36 amino acids) protein. Each subunit contains a high-spin iron atom tetrahedrally bound to four cysteinyl sulfur atoms, a metal center similar to that found in rubredoxin (Rd) type proteins. The simplicity of the active center in Dx and the possibility of replacing the iron by other metals make this protein an attractive case for the crystallographic analysis of metal-substituted derivatives. This study extends the relevance of Dx to the bioinorganic chemistry field and is important to obtain model compounds that can mimic the four sulfur coordination of metals in biology. Metal replacement experiments were carried out by reconstituting the apoprotein with In3+, Ga3+, Cd2+, Hg2+, and Ni2+ salts. The In3+ and Ga3+ derivatives are isomorphous with the iron native protein; whereas Cd2+, Hg2+, and Ni2+ substituted Dx crystallized under different experimental conditions, yielding two additional crystal morphologies; their structures were determined by the molecular replacement method. A comparison of the three-dimensional structures for all metal derivatives shows that the overall secondary and tertiary structures are maintained, while some differences in metal coordination geometry occur, namely, bond lengths and angles of the metal with the sulfur ligands. These data are discussed in terms of the entatic state theory.

Amino Acid Sequence↗
PubMed1998

Altered specificity mutations define residues essential for substrate positioning in xanthine dehydrogenase.

We describe the sequence changes of a number of mutations of the Aspergillus nidulans xanthine dehydrogenase (XDH). We have located the amino acids affected by these changes in the three-dimensional (3D) structure of aldehyde oxido-reductase (MOP) from Desulfovibrio gigas, related to eukaryotic XDHs. Of these, two are loss of function mutations, mapping, respectively, in the molybdenum-pterin co-factor (MoCo) domain and in the domain involved in substrate recognition. Changes in two amino acids result in resistance to the irreversible inhibitor allopurinol. In Arg911 two different changes, conserved among all XDHs and MOP but not in other aldehyde oxidases (AO), change the position of hydroxylation of the analogue 2-hydroxypurine from C-8 to C-6. A number of changes affect residues adjacent to the molybdenum or its ligands. Arg911 is positioned in the substrate pocket in a way that it can account for the positioning of purine substrates in relation to the MoCo reactive center, together with a glutamate residue, universally conserved among the XDHs (Glu833).

Alleles↗
PubMed1997

Crystal structure of acidic seminal fluid protein (aSFP) at 1.9 A resolution: a bovine polypeptide of the spermadhesin family.

We report the three-dimensional crystal structure of acidic seminal fluid protein (aSFP), a 12.9 kDa polypeptide of the spermadhesin family isolated from bovine seminal plasma, solved by the multiple isomorphous replacement method and refined with data to 1.9 A resolution with a final R-factor of 17.3%. aSFP is built by a single CUB domain architecture, a 100 to 110 amino-acid-residue extracellular module found in 16 functionally diverse proteins. The structure of aSFP reveals that the CUB domain displays a beta-sandwich topology organised into two 5-stranded beta-sheets, each of which contain two parallel and four antiparallel strands. The structure of aSFP is almost identical to that of porcine spermadhesins PSP-I and PSP-II, which in turn show limited structural similarity with jellyroll topologies of certain virus capsid proteins. Essentially, topologically conserved residues in these proteins are those internal amino acids forming the hydrophobic core of the CUB and the jellyroll domains, suggesting their importance in maintaining the integrity of these protein folds. On the other hand, the structure of aSFP shows structural features that are unique to this protein and which may provide a structural ground for understanding the distinct biological properties of different members of the spermadhesin protein family.

Amino Acid Sequence↗
PubMed1997

The 2.4 A resolution crystal structure of boar seminal plasma PSP-I/PSP-II: a zona pellucida-binding glycoprotein heterodimer of the spermadhesin family built by a CUB domain architecture.

The crystal structure of porcine seminal plasma spermadhesin PSP-I/PSP-II heterodimer has been determined in two crystal forms by multiple isomorphous replacement in an hexagonal crystal (space group P6(1)22) and molecular replacement in a trigonal crystal of space group P3(2)21. The crystal structure has been refined at 2.4 A resolution to an R-factor of 20.0% (Rfree = 25.9%) for 14,809 independent reflections with intensities greater than 2 sigma (I), with root-mean-square deviations of 0.009 A and 1.657 degrees from ideal bond lengths and bond angles, respectively. The final model includes 1688 non-hydrogen protein atoms of 221 amino acids and 79 water molecules. PSP-I/PSP-II represents the first crystal structure of a mammalian zona pellucida-binding protein. PSP-II displays a putative carbohydrate-recognition site located around its Asn50. This region shares structural features with sugar binding sites of known lectin structures of the leguminous and galectin families. PSP-I and PSP-II are N-glycosylated at asparagine residues 50 and 98, respectively, and show site heterogeneity. Only the innermost N-acetylglucosamine of PSP-I is defined in the crystal structure. Both subunits of the PSP-I/PSP-II heterodimer are built by a single CUB domain architecture. The CUB domain displays a novel fold, which consists of a compact ellipsoidal beta-sandwich structure (42 A x 27 A x 23 A) organized into two 5-stranded beta-sheets. Each sheet contains parallel and antiparallel beta-strands. Two disulphide bridges, which are conserved in all spermadhesin molecules and many CUB domains, crosslink loop LA and strand beta 4 and loops LE and LG, respectively, at opposite edges of the same face of the domain. The four highly conserved aromatic residues and 15 out of 17 invariant hydrophobic residues, which define the CUB domain signature, display an interior location, suggesting that this hydrophobic core may be essential for maintaining the overall folding of the domain. Most of the hydrophobic core residue characteristics are conserved in the jellyroll topology of certain icosahedral virus capsid proteins, indicating that the CUB domain and the viral proteins share a minimal structural core.

Amino Acid Sequence↗
PubMed1997

Crystallization and preliminary X-ray diffraction studies of aSFP, a bovine seminal plasma protein with a single CUB domain architecture.

Bovine acidic seminal fluid protein (aSFP) is a 1.29 kDa polypeptide of the spermadhesin family built by a single CUB domain architecture. The CUB domain is an extracellular module present in 16 functionally diverse proteins. To determine the three-dimensional structure of aSFP, the protein was crystallized at 21 degrees C by vapor diffusion in hanging drops, using ammonium sulfate, pH 4.7, and polyethyleneglycol 4,000 as precipitants, containing 10% dioxane to avoid the formation of clustered crystals. Elongated prismatic crystals with maximal size of 0.6 x 0.3 x 0.2 mm3 diffract to beyond 1.9 A resolution and belong to space group P2(1)2(1)2(1), with cell parameters a = 52.4 A, b = 41.5 A, c = 48.2 A. There is one aSFP molecule per asymmetric unit, which corresponds to a crystal volume per unit molecular mass of 2.04 A3/Da, and analytical ultracentrifugation analysis show that aSFP is a monomeric protein.

Animals↗
PubMed1997

Structure and function of molybdopterin containing enzymes.

Molybdopterin containing enzymes are present in a wide range of living systems and have been known for several decades. However, only in the past two years have the first crystal structures been reported for this type of enzyme. This has represented a major breakthrough in this field. The enzymes share common structural features, but reveal different polypeptide folding topologies. In this review we give an account of the related spectroscopic information and the crystallographic results, with emphasis on structure-function studies.

Coenzymes↗
PubMed1997

The crystal structures of two spermadhesins reveal the CUB domain fold.

Spermadhesins, 12,000-14,000 M(r) mammalian proteins, include lectins involved in sperm-egg binding and display a single CUB domain architecture. We report the crystal structures of porcine seminal plasma PSP-I/PSP-II, a heterodimer of two glycosylated spermadhesins, and bovine aSFP at 2.4 A and 1.9 A resolution respectively.

Amino Acid Sequence↗