Biomedical subjects

Ignacio Fita

Publications and source records attributed to Ignacio Fita.

32 records · Page 2Linked to original sources

PubMed2003

Characterization of the catalase-peroxidase KatG from Burkholderia pseudomallei by mass spectrometry.

The electron density maps of the catalase-peroxidase from Burkholderia pseudomallei (BpKatG) presented two unusual covalent modifications. A covalent structure linked the active site Trp111 with Tyr238 and Tyr238 with Met264, and the heme was modified, likely by a perhydroxy group added to the vinyl group on ring I. Mass spectrometry analysis of tryptic digests of BpKatG revealed a cluster of ions at m/z 6585, consistent with the fusion of three peptides through Trp111, Tyr238, and Met264, and a cluster at m/z approximately 4525, consistent with the fusion of two peptides linked through Trp111 and Tyr238. MS/MS analysis of the major ions at m/z 4524 and 4540 confirmed the expected sequence and suggested that the multiple ions in the cluster were the result of multiple oxidation events and transfer of CH3-S to the tyrosine. Neither cluster of ions at m/z 4525 or 6585 was present in the spectrum of a tryptic digest of the W111F variant of BpKatG. The spectrum of the tryptic digest of native BpKatG also contained a major ion for a peptide in which Met264 had been converted to homoserine, consistent with the covalent bond between Tyr238 and Met264 being susceptible to hydrolysis, including the loss of the CH3-S from the methionine. Analysis of the tryptic digests of hydroperoxidase I (KatG) from Escherichia coli provided direct evidence for the covalent linkage between Trp105 and Tyr226 and indirect evidence for a covalent linkage between Tyr226 and Met252. Tryptic peptide analysis and N-terminal sequencing revealed that the N-terminal residue of BpKatG is Ser22.

Amino Acid Sequence↗

PubMed2003

Crystal structure of the vertebrate NADP(H)-dependent alcohol dehydrogenase (ADH8).

The amphibian enzyme ADH8, previously named class IV-like, is the only known vertebrate alcohol dehydrogenase (ADH) with specificity towards NADP(H). The three-dimensional structures of ADH8 and of the binary complex ADH8-NADP(+) have been now determined and refined to resolutions of 2.2A and 1.8A, respectively. The coenzyme and substrate specificity of ADH8, that has 50-65% sequence identity with vertebrate NAD(H)-dependent ADHs, suggest a role in aldehyde reduction probably as a retinal reductase. The large volume of the substrate-binding pocket can explain both the high catalytic efficiency of ADH8 with retinoids and the high K(m) value for ethanol. Preference of NADP(H) appears to be achieved by the presence in ADH8 of the triad Gly223-Thr224-His225 and the recruitment of conserved Lys228, which define a binding pocket for the terminal phosphate group of the cofactor. NADP(H) binds to ADH8 in an extended conformation that superimposes well with the NAD(H) molecules found in NAD(H)-dependent ADH complexes. No additional reshaping of the dinucleotide-binding site is observed which explains why NAD(H) can also be used as a cofactor by ADH8. The structural features support the classification of ADH8 as an independent ADH class.

Alcohol Dehydrogenase↗

PubMed2003

An electrical potential in the access channel of catalases enhances catalysis.

Substrate H2O2 must gain access to the deeply buried active site of catalases through channels of 30-50 A in length. The most prominent or main channel approaches the active site perpendicular to the plane of the heme and contains a number of residues that are conserved in all catalases. Changes in Val169, 8 A from the heme in catalase HPII from Escherichia coli, introducing smaller, larger or polar side chains reduces the catalase activity. Changes in Asp181, 12 A from the heme, reduces activity by up to 90% if the negatively charged side chain is removed when Ala, Gln, Ser, Asn, or Ile are the substituted residues. Only the D181E variant retains wild type activity. Determination of the crystal structures of the Glu181, Ala181, Ser181, and Gln181 variants of HPII reveals lower water occupancy in the main channel of the less active variants, particularly at the position forming the sixth ligand to the heme iron and in the hydrophobic, constricted region adjacent to Val169. It is proposed that an electrical potential exists between the negatively charged aspartate (or glutamate) side chain at position 181 and the positively charged heme iron 12 A distant. The potential field acts upon the electrical dipoles of water generating a common orientation that favors hydrogen bond formation and promotes interaction with the heme iron. Substrate hydrogen peroxide would be affected similarly and would enter the active site oriented optimally for interaction with active site residues.

Arginine↗

PubMed2003

Catalase-peroxidase KatG of Burkholderia pseudomallei at 1.7A resolution.

The catalase-peroxidase encoded by katG of Burkholderia pseudomallei (BpKatG) is 65% identical with KatG of Mycobacterium tuberculosis, the enzyme responsible for the activation of isoniazid as an antibiotic. The structure of a complex of BpKatG with an unidentified ligand, has been solved and refined at 1.7A resolution using X-ray synchrotron data collected from crystals flash-cooled with liquid nitrogen. The crystallographic agreement factors R and R(free) are 15.3% and 18.6%, respectively. The crystallized enzyme is a dimer with one modified heme group and one metal ion, likely sodium, per subunit. The modification on the heme group involves the covalent addition of two or three atoms, likely a perhydroxy group, to the secondary carbon atom of the vinyl group on ring I. The added group can form hydrogen bonds with two water molecules that are also in contact with the active-site residues Trp111 and His112, suggesting that the modification may have a catalytic role. The heme modification is in close proximity to an unusual covalent adduct among the side-chains of Trp111, Tyr238 and Met264. In addition, Trp111 appears to be oxidized on C(delta1) of the indole ring. The main channel, providing access of substrate hydrogen peroxide to the heme, contains a region of unassigned electron density consistent with the binding of a pyridine nucleotide-like molecule. An interior cavity, containing the sodium ion and an additional region of unassigned density, is evident adjacent to the adduct and is accessible to the outside through a second funnel-shaped channel. A large cleft in the side of the subunit is evident and may be a potential substrate-binding site with a clear pathway for electron transfer to the active-site heme group through the adduct.

Antitubercular Agents↗

PubMed2003

Crystallization and preliminary X-ray analysis of swine vesicular disease virus (SVDV).

Three different crystal forms of the swine vesicular disease virus (SVDV), isolate SPA/2/'93, were obtained by the hanging-drop vapour-diffusion technique using ammonium sulfate and sodium/potassium phosphate as precipitants. Monoclinic crystals, space group C2, with unit-cell parameters a = 473.7, b = 385.3, c = 472.8 A, beta = 100.4 degrees, contain one virus pArticle in the crystal asymmetric unit and diffract to 3.0 A resolution. A second type of crystals had a cubic morphology and diffracted beyond 2.6 A resolution. These crystals belong to a primitive orthorhombic space group, with unit-cell parameters a = 319.6, b = 353.8, c = 377.7 A, and contain half a virus pArticle in the asymmetric unit. A third type of crystals, with a prismatic shape and belonging to space group I222, was also obtained under similar crystallization conditions. These latter crystals, with unit-cell parameters a = 318.3, b = 349.9, c = 371.7 A, diffract to at least 3.0 A resolution and contain 15 protomers per asymmetric unit; this requires that three perpendicular crystal twofold axes coincide with three of the viral pArticles' dyad axes.

Ammonium Sulfate↗

PubMed2003

Structure of the Clade 1 catalase, CatF of Pseudomonas syringae, at 1.8 A resolution.

Catalase CatF of Pseudomonas syringae has been identified phylogenetically as a clade 1 catalase, closely related to plant catalases, a group from which no structure has been determined. The structure of CatF has been refined at 1.8 A resolution by using X-ray synchrotron data collected from a crystal flash-cooled with liquid nitrogen. The crystallographic agreement factors R and R(free) are, respectively, 18.3% and 24.0%. The asymmetric unit of the crystal contains a whole molecule that shows accurate 222-point group symmetry. The crystallized enzyme is a homotetramer of subunits with 484 residues, some 26 residues shorter than predicted from the DNA sequence. Mass spectrometry analysis confirmed the absence of 26 N-terminal residues, possibly removed by a periplasmic transport system. The core structure of the CatF subunit was closely related to seven other catalases with root-mean-square deviations (RMSDs) of 368 core Calpha atoms of 0.99-1.30 A. The heme component of CatF is heme b in the same orientation that is found in Escherichia coli hydroperoxidase II, an orientation that is flipped 180 degrees with respect the orientation of the heme in bovine liver catalase. NADPH is not found in the structure of CatF because key residues required for nucleotide binding are missing; 2129 water molecules were refined into the model. Water occupancy in the main or perpendicular channel of CatF varied among the four subunits from two to five in the region between the heme and the conserved Asp150. A comparison of the water occupancy in this region with the same region in other catalases reveals significant differences among the catalases.

Binding Sites↗

PubMed2003

Crystallization and preliminary X-ray analysis of NADP(H)-dependent alcohol dehydrogenases from Saccharomyces cerevisiae and Rana perezi.

Different crystal forms diffracting to high resolution have been obtained for two NADP(H)-dependent alcohol dehydrogenases, members of the medium-chain dehydrogenase/reductase superfamily: ScADHVI from Saccharomyces cerevisiae and ADH8 from Rana perezi. ScADHVI is a broad-specificity enzyme, with a sequence identity lower than 25% with respect to all other ADHs of known structure. The best crystals of ScADHVI diffracted beyond 2.8 A resolution and belonged to the trigonal space group P3(1)21 (or to its enantiomorph P3(2)21), with unit-cell parameters a = b = 102.2, c = 149.7 A, gamma = 120 degrees. These crystals were produced by the hanging-drop vapour-diffusion method using ammonium sulfate as precipitant. Packing considerations together with the self-rotation function and the native Patterson map seem to indicate the presence of only one subunit per asymmetric unit, with a Volume solvent content of about 80%. ADH8 from R. perezi is the only NADP(H)-dependent ADH from vertebrates characterized to date. Crystals of ADH8 obtained both in the absence and in the presence of NADP(+) using polyethylene glycol and lithium sulfate as precipitants diffracted to 2.2 and 1.8 A, respectively, using synchrotron radiation. These crystals were isomorphous, space group C2, with approximate unit-cell parameters a = 122, b = 79, c = 91 A, beta = 113 degrees and contain one dimer per asymmetric unit, with a Volume solvent content of about 50%.

Alcohol Dehydrogenase↗

PubMed2003

Structure of swine vesicular disease virus: mapping of changes occurring during adaptation of human coxsackie B5 virus to infect swine.

The structure of swine vesicular disease virus (SVDV) was solved and refined at a 3.0-A resolution by X-ray crystallography to gain information about the role of sequence changes that occurred as this virus evolved from the parental human pathogen coxsackievirus B5 (CVB5). These amino acid substitutions can be clustered in five distinct regions: (i) the antigenic sites, (ii) the hydrophobic pocket of the VP1 beta-sandwich, (iii) the putative CAR binding site, (iv) the putative heparan sulfate binding site, and (v) the fivefold axis. The VP1 pocket is occupied by a branched pocket factor, apparently different from that present in the closely related virus CVB3 and in other picornaviruses. This finding may be relevant for the design of new antiviral compounds against this site. Density consistent with the presence of ions was observed on the fivefold and threefold axes. The structure also provided an accurate description of the putative receptor binding sites.

Adaptation, Physiological↗

PubMed2002

Crystallization and preliminary X-ray analysis of the catalase-peroxidase KatG from Burkholderia pseudomallei.

The bifunctional catalase-peroxidase KatG encoded by the katG gene of Burkholderia pseudomallei has a predicted subunit size of 81.6 kDa. It shows high sequence similarity to other catalase-peroxidases of bacterial, archaebacterial and fungal origin, including 64% identity to KatG from Mycobacterium tuberculosis and lesser sequence similarity to members of the plant peroxidase family. Crystals from this protein were grown in 16-20% PEG 4000, 20% 2-methyl-2,4-pentanediol and 0.1 M sodium citrate pH 5.6 by the hanging-drop vapour-diffusion method at 293 K. These crystals diffracted beyond 1.8 A resolution and belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 100.9, b = 115.6, c = 175.2 A. The data are consistent with either a monomer or a dimer in the crystal asymmetric unit.

Bacterial Proteins↗

PubMed2002

A crystallographic glimpse of a nucleotide triphosphate (AMPPNP) bound to a protein surface: external and internal AMPPNP molecules in crystalline N-acetyl-L-glutamate kinase.

A large volume of unexplained electron density in a crystal of N-acetyl-L-glutamate kinase (NAGK) is now interpreted as an external, very extended, metal-free AMPPNP molecule that occupies two alternative positions and that makes contacts with the protein exclusively through its gamma-imidophosphate. This external nucleotide is compared with the active-site nucleotide and the reasons for its extended shape, lack of complexed metal and peripheral binding are analyzed. Further, the possibility that this bystander AMPPNP is waiting to occupy the active centre is discussed.

Adenylyl Imidodiphosphate↗

PubMed2002

Additional binding sites for anionic phospholipids and calcium ions in the crystal structures of complexes of the C2 domain of protein kinase calpha.

The C2 domain of protein kinase Calpha (PKCalpha) corresponds to the regulatory sequence motif, found in a large variety of membrane trafficking and signal transduction proteins, that mediates the recruitment of proteins by phospholipid membranes. In the PKCalpha isoenzyme, the Ca2+-dependent binding to membranes is highly specific to 1,2-sn-phosphatidyl-l-serine. Intrinsic Ca2+ binding tends to be of low affinity and non-cooperative, while phospholipid membranes enhance the overall affinity of Ca2+ and convert it into cooperative binding. The crystal structure of a ternary complex of the PKCalpha-C2 domain showed the binding of two calcium ions and of one 1,2-dicaproyl-sn-phosphatidyl-l-serine (DCPS) molecule that was coordinated directly to one of the calcium ions. The structures of the C2 domain of PKCalpha crystallised in the presence of Ca2+ with either 1,2-diacetyl-sn-phosphatidyl-l-serine (DAPS) or 1,2-dicaproyl-sn-phosphatidic acid (DCPA) have now been determined and refined at 1.9 A and at 2.0 A, respectively. DAPS, a phospholipid with short hydrocarbon chains, was expected to facilitate the accommodation of the phospholipid ligand inside the Ca2+-binding pocket. DCPA, with a phosphatidic acid (PA) head group, was used to investigate the preference for phospholipids with phosphatidyl-l-serine (PS) head groups. The two structures determined show the presence of an additional binding site for anionic phospholipids in the vicinity of the conserved lysine-rich cluster. Site-directed mutagenesis, on the lysine residues from this cluster that interact directly with the phospholipid, revealed a substantial decrease in C2 domain binding to vesicles when concentrations of either PS or PA were increased in the absence of Ca2+. In the complex of the C2 domain with DAPS a third Ca2+, which binds an extra phosphate group, was identified in the calcium-binding regions (CBRs). The interplay between calcium ions and phosphate groups or phospholipid molecules in the C2 domain of PKCalpha is supported by the specificity and spatial organisation of the binding sites in the domain and by the variable occupancies of ligands found in the different crystal structures. Implications for PKCalpha activity of these structural results, in particular at the level of the binding affinity of the C2 domain to membranes, are discussed.

Animals↗

PubMed2002

Towards structural understanding of feedback control of arginine biosynthesis: cloning and expression of the gene for the arginine-inhibited N-acetyl-L-glutamate kinase from Pseudomonas aeruginosa, purification and crystallization of the recombinant enzyme and preliminary X-ray studies.

N-Acetyl-L-glutamate kinase (NAGK) catalyzes the second step in the pathway of arginine biosynthesis in microorganisms and plants. In many species, it is the pathway-controlling enzyme and is subject to feedback inhibition by arginine. The gene for the best characterized arginine-inhibitable NAGK, that from Pseudomonas aeruginosa, has been cloned in a pET22 plasmid and overexpressed in Escherichia coli. The enzyme was purified in three steps to 95% purity and was shown by cross-linking to form dimers. It was crystallized by the hanging-drop vapour-diffusion method at 277 K in the presence of ADP, Mg and N-acetyl-L-glutamate. The crystallization solution contained 0.1 M sodium cacodylate pH 6.5, 150-170 mM magnesium acetate and 13% polyethylene glycol 8000. Prismatic crystals of maximum dimension approximately 0.5 mm diffract to 2.75 A resolution and belong to space group P1 (unit-cell parameters a = 71.86, b = 98.78, c = 162.9 A, alpha = 91.49, beta = 92.03, gamma = 107.56 degrees ). Packing density considerations agree with 6-18 NAGK monomers in the asymmetric unit, with a corresponding solvent content of 79-36%. Self-rotation function calculations confirm the space group and suggest the presence of 3-7 dimers in the unit cell.

Arginine↗

PubMed2002

Crystallization and preliminary X-ray analysis of the hydroperoxidase I C-terminal domain from Escherichia coli.

Hydroperoxidases (HP) are normally large haem-containing bifunctional enzymes capable of acting as both catalases and peroxidases. The C-terminal domain of HPI from Escherichia coli (KatG), extending from residue Tyr422 to Leu726, was found to be resistant to trypsin proteolysis. The segment of katG encoding this domain was cloned and overexpressed to produce a haemless protein that is soluble even at concentrations above 30 mg ml(-1). This protein shows a 25% sequence identity with cytochrome c peroxidase (CCP) from Saccharomyces cerevisae, despite lacking the characteristic catalytic and iron-binding residues. Crystals from this protein were grown in 0.6 M sodium citrate buffered to pH 7.5 with HEPES by the hanging-drop vapour-diffusion method at 293 K. These crystals diffracted beyond 2.0 A resolution and belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 84.2, b = 98.7, c = 302.8 A. Three pseudo-origin peaks in the Patterson maps indicate an unusual packing compatible with the presence of three molecules in the crystal asymmetric unit and a solvent content of about 80% by volume.

Catalase↗

PubMed2002

Structure of acetylglutamate kinase, a key enzyme for arginine biosynthesis and a prototype for the amino acid kinase enzyme family, during catalysis.

N-Acetyl-L-glutamate kinase (NAGK), a member of the amino acid kinase family, catalyzes the second and frequently controlling step of arginine synthesis. The Escherichia coli NAGK crystal structure to 1.5 A resolution reveals a 258-residue subunit homodimer nucleated by a central 16-stranded molecular open beta sheet sandwiched between alpha helices. In each subunit, AMPPNP, as an alphabetagamma-phosphate-Mg2+ complex, binds along the sheet C edge, and N-acetyl-L-glutamate binds near the dyadic axis with its gamma-COO- aligned at short distance from the gamma-phosphoryl, indicating associative phosphoryl transfer assisted by: (1) Mg2+ complexation; (2) the positive charges on Lys8, Lys217, and on two helix dipoles; and (3) by hydrogen bonding with the y-phosphate. The structural resemblance with carbamate kinase and the alignment of the sequences suggest that NAGK is a structural and functional prototype for the amino acid kinase family, which differs from other acylphosphate-making devices represented by phosphoglycerate kinase, acetate kinase, and biotin carboxylase.

Amino Acid Sequence↗