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Biomedical subjects

A Henriksen

Publications and source records attributed to A Henriksen.

At least 19 recordsLinked to original sources
PubMed2001

Major venom allergen of yellow jackets, Ves v 5: structural characterization of a pathogenesis-related protein superfamily.

Ves v 5 is one of three major allergens found in yellow-jacket venom: phospholipase A(1) (Ves v 1), hyaluronidase (Ves v 2), and antigen 5 (Ves v 5). Ves v 5 is related by high amino acid sequence identity to pathogenesis-related proteins including proteins from mammals, reptiles, insects, fungi, and plants. The crystal structure of Ves v 5 has been solved and refined to a resolution of 1.9 A. The majority of residues conserved between the pathogenesis-related proteins can be rationalized in terms of hydrogen bonding patterns and hydrophobic interactions defining an alpha-beta-alpha sandwich core structure. A small number of consensus residues are solvent exposed (including two adjacent histidines) and located in an elongated cavity that forms a putative active site. The site has no structural resemblance to previously characterized enzymes. Homologous antigen 5's from a large number of different yellow jackets, hornets, and paper wasps are known and patients show varying extents of cross-reactivity to the related antigen 5's. The structure of Ves v 5 allows a detailed analysis of the epitopes that may participate in antigenic cross-reactivity, findings that are useful for the development of a vaccine for treatment of insect allergy.

Allergens↗
PubMed2001

Shedding light on disulfide bond formation: engineering a redox switch in green fluorescent protein.

To visualize the formation of disulfide bonds in living cells, a pair of redox-active cysteines was introduced into the yellow fluorescent variant of green fluorescent protein. Formation of a disulfide bond between the two cysteines was fully reversible and resulted in a >2-fold decrease in the intrinsic fluorescence. Inter conversion between the two redox states could thus be followed in vitro as well as in vivo by non-invasive fluorimetric measurements. The 1.5 A crystal structure of the oxidized protein revealed a disulfide bond-induced distortion of the beta-barrel, as well as a structural reorganization of residues in the immediate chromophore environment. By combining this information with spectroscopic data, we propose a detailed mechanism accounting for the observed redox state-dependent fluorescence. The redox potential of the cysteine couple was found to be within the physiological range for redox-active cysteines. In the cytoplasm of Escherichia coli, the protein was a sensitive probe for the redox changes that occur upon disruption of the thioredoxin reductive pathway.

Crystallography, X-Ray↗
PubMed2001

Differential activity and structure of highly similar peroxidases. Spectroscopic, crystallographic, and enzymatic analyses of lignifying Arabidopsis thaliana peroxidase A2 and horseradish peroxidase A2.

Anionic Arabidopsis thaliana peroxidase ATP A2 was expressed in Escherichia coli and used as a model for the 95% identical commercially available horseradish peroxidase HRP A2. The crystal structure of ATP A2 at 1.45 A resolution at 100 K showed a water molecule only 2.1 A from heme iron [Ostergaard, L., et al. (2000) Plant Mol. Biol. 44, 231-243], whereas spectroscopic studies of HRP A2 in solution at room temperature [Feis, A., et al. (1998) J. Raman Spectrosc. 29, 933-938] showed five-coordinated heme iron, which is common in peroxidases. Presented here, the X-ray crystallographic, single-crystal, and solution resonance Raman studies at room temperature confirmed that the sixth coordination position of heme iron of ATP A2 is essentially vacant. Furthermore, electronic absorption and resonance Raman spectroscopy showed that the heme environments of recombinant ATP A2 and glycosylated plant HRP A2 are indistinguishable at neutral and alkaline pH, from room temperature to 12 K, and are highly flexible compared with other plant peroxidases. Ostergaard et al. (2000) also demonstrated that ATP A2 expression and lignin formation coincide in Arabidopsis tissues, and docking of lignin precursors into the substrate binding site of ATP A2 predicted that coniferyl and p-coumaryl alcohols were good substrates. In contrast, the additional methoxy group of the sinapyl moiety gave rise to steric hindrance, not only in A2 type peroxidases but also in all peroxidases. We confirm these predictions for ATP A2, HRP A2, and HRP C. The specific activity of ATP A2 was lower than that of HRP A2 (pH 4-8), although a steady-state study at pH 5 demonstrated very little difference in their rate constants for reaction with H2O2 (k1 = 1.0 microM(-1) x s(-1). The oxidation of coniferyl alcohol, ferulic, p-coumaric, and sinapic acids by HRP A2, and ATP A2, however, gave modest but significantly different k3 rate constants of 8.7 +/- 0.3, 4.0 +/- 0.2, 0.70 +/- 0.03, and 0.04 +/- 0.2 microM(-1) x s(-1) for HRP A2, respectively, and 4.6 +/- 0.2, 2.3 +/- 0.1, 0.25 +/- 0.01, and 0.01 +/- 0.004 microM(-1) x s(-1) for ATP A2, respectively. The structural origin of the differential reactivity is discussed in relation to glycosylation and amino acid substitutions. The results are of general importance to the use of homologous models and structure determination at low temperatures.

Arabidopsis↗
PubMed2001

Amylosucrase, a glucan-synthesizing enzyme from the alpha-amylase family.

Amylosucrase (E.C. 2.4.1.4) is a member of Family 13 of the glycoside hydrolases (the alpha-amylases), although its biological function is the synthesis of amylose-like polymers from sucrose. The structure of amylosucrase from Neisseria polysaccharea is divided into five domains: an all helical N-terminal domain that is not similar to any known fold, a (beta/alpha)(8)-barrel A-domain, B- and B'-domains displaying alpha/beta-structure, and a C-terminal eight-stranded beta-sheet domain. In contrast to other Family 13 hydrolases that have the active site in the bottom of a large cleft, the active site of amylosucrase is at the bottom of a pocket at the molecular surface. A substrate binding site resembling the amylase 2 subsite is not found in amylosucrase. The site is blocked by a salt bridge between residues in the second and eight loops of the (beta/alpha)(8)-barrel. The result is an exo-acting enzyme. Loop 7 in the amylosucrase barrel is prolonged compared with the loop structure found in other hydrolases, and this insertion (forming domain B') is suggested to be important for the polymer synthase activity of the enzyme. The topology of the B'-domain creates an active site entrance with several ravines in the molecular surface that could be used specifically by the substrates/products (sucrose, glucan polymer, and fructose) that have to get in and out of the active site pocket.

Amino Acid Sequence↗
PubMed2001

Structure of soybean seed coat peroxidase: a plant peroxidase with unusual stability and haem-apoprotein interactions.

Soybean seed coat peroxidase (SBP) is a peroxidase with extraordinary stability and catalytic properties. It belongs to the family of class III plant peroxidases that can oxidize a wide variety of organic and inorganic substrates using hydrogen peroxide. Because the plant enzyme is a heterogeneous glycoprotein, SBP was produced recombinant in Escherichia coli for the present crystallographic study. The three-dimensional structure of SBP shows a bound tris(hydroxymethyl)aminomethane molecule (TRIS). This TRIS molecule has hydrogen bonds to active site residues corresponding to the residues that interact with the small phenolic substrate ferulic acid in the horseradish peroxidase C (HRPC):ferulic acid complex. TRIS is positioned in what has been described as a secondary substrate-binding site in HRPC, and the structure of the SBP:TRIS complex indicates that this secondary substrate-binding site could be of functional importance. SBP has one of the most solvent accessible delta-meso haem edge (the site of electron transfer from reducing substrates to the enzymatic intermediates compound I and II) so far described for a plant peroxidase and structural alignment suggests that the volume of Ile74 is a factor that influences the solvent accessibility of this important site. A contact between haem C8 vinyl and the sulphur atom of Met37 is observed in the SBP structure. This interaction might affect the stability of the haem group by stabilisation/delocalisation of the porphyrin pi-cation of compound I.

Binding Sites↗
PubMed2000

Dominant epitopes and allergic cross-reactivity: complex formation between a Fab fragment of a monoclonal murine IgG antibody and the major allergen from birch pollen Bet v 1.

The symptoms characteristic of allergic hypersensitivity are caused by the release of mediators, i.e., histamine, from effector cells such as basophils and mast cells. Allergens with more than one B cell epitope cross-link IgE Abs bound to high affinity FcepsilonRI receptors on mast cell surfaces leading to aggregation and subsequent mediator release. Thus, allergen-Ab complexes play a crucial role in the cascade leading to the allergic response. We here report the structure of a 1:1 complex between the major birch pollen allergen Bet v 1 and the Fab fragment from a murine monoclonal IgG1 Ab, BV16, that has been solved to 2.9 A resolution by x-ray diffraction. The mAb is shown to inhibit the binding of allergic patients' IgE to Bet v 1, and the allergen-IgG complex may therefore serve as a model for the study of allergen-IgE interactions relevant in allergy. The size of the BV16 epitope is 931 A2 as defined by the Bet v 1 Ab interaction surface. Molecular interactions predicted to occur in the interface are likewise in agreement with earlier observations on Ag-Ab complexes. The epitope is formed by amino acids that are conserved among major allergens from related species within the Fagales order. In combination with a surprisingly high inhibitory capacity of BV16 with respect to allergic patients' serum IgE binding to Bet v 1, these observations provide experimental support for the proposal of dominant IgE epitopes located in the conserved surface areas. This model will facilitate the development of new and safer vaccines for allergen immunotherapy in the form of mutated allergens.

Allergens↗
PubMed2000

Arabidopsis ATP A2 peroxidase. Expression and high-resolution structure of a plant peroxidase with implications for lignification.

Lignins are phenolic biopolymers synthesized by terrestrial, vascular plants for mechanical support and in response to pathogen attack. Peroxidases have been proposed to catalyse the dehydrogenative polymerization of monolignols into lignins, although no specific isoenzyme has been shown to be involved in lignin biosynthesis. Recently we isolated an extracellular anionic peroxidase, ATP A2, from rapidly lignifying Arabidopsis cell suspension culture and cloned its cDNA. Here we show that the Atp A2 promoter directs GUS reporter gene expression in lignified tissues of transgenic plants. Moreover, an Arabidopsis mutant with increased lignin levels compared to wild type shows increased levels of ATP A2 mRNA and of a mRNA encoding an enzyme upstream in the lignin biosynthetic pathway. The substrate specificity of ATP A2 was analysed by X-ray crystallography and docking of lignin precursors. The structure of ATP A2 was solved to 1.45 A resolution at 100 K. Docking of p-coumaryl, coniferyl and sinapyl alcohol in the substrate binding site of ATP A2 were analysed on the basis of the crystal structure of a horseradish peroxidase C-CN-ferulic acid complex. The analysis indicates that the precursors p-coumaryl and coniferyl alcohols are preferred by ATP A2, while the oxidation of sinapyl alcohol will be sterically hindered in ATP A2 as well as in all other plant peroxidases due to an overlap with the conserved Pro-139. We suggest ATP A2 is involved in a complex regulation of the covalent cross-linking in the plant cell wall.

Arabidopsis↗
PubMed2000

Crystallization and preliminary X-ray studies of recombinant amylosucrase from Neisseria polysaccharea.

Recombinant amylosucrase from Neisseria polysaccharea was crystallized by the vapour-diffusion procedure in the presence of polyethylene glycol 6000. The crystals belong to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 95.7, b = 117.2, c = 62.1 A, and diffract to 1.6 A resolution. A p-chloromercuribenzene sulfonate (pcmbs) derivative has been identified and a selenomethionine-substituted protein has been produced and crystallized.

Bacterial Proteins↗
PubMed2000

Arabidopsis thaliana peroxidase N: structure of a novel neutral peroxidase.

The structure of the neutral peroxidase from Arabidopsis thaliana (ATP N) has been determined to a resolution of 1.9 A and a free R value of 20.5%. ATP N has the expected characteristic fold of the class III peroxidases, with a C(alpha) r.m.s.d. of 0.82 A when compared with horseradish peroxidase C (HRP C). HRP C is 54% identical to ATP N in sequence. When the structures of four class III plant peroxidases are superimposed, the regions with structural differences are non-randomly distributed; all are located in one half of the molecule. The architecture of the haem pocket of ATP N is very similar to that of HRP C, in agreement with the low small-molecule substrate specificity of all class III peroxidases. The structure of ATP N suggests that the pH dependence of the substrate turnover will differ from that of HRP C owing to differences in polarity of the residues in the substrate-access channel. Since there are fewer hydrogen bonds to haem C17 propionate O atoms in ATP N than in HRP C, it is suggested that ATP N will lose haem more easily than HRP C. Unlike almost all other class III plant peroxidases, ATP N has a free cysteine residue at a similar position to the suggested secondary substrate-binding site in lignin peroxidase.

Amino Acid Sequence↗
PubMed1999

The structures of the horseradish peroxidase C-ferulic acid complex and the ternary complex with cyanide suggest how peroxidases oxidize small phenolic substrates.

We have solved the x-ray structures of the binary horseradish peroxidase C-ferulic acid complex and the ternary horseradish peroxidase C-cyanide-ferulic acid complex to 2.0 and 1.45 A, respectively. Ferulic acid is a naturally occurring phenolic compound found in the plant cell wall and is an in vivo substrate for plant peroxidases. The x-ray structures demonstrate the flexibility and dynamic character of the aromatic donor binding site in horseradish peroxidase and emphasize the role of the distal arginine (Arg(38)) in both substrate oxidation and ligand binding. Arg(38) hydrogen bonds to bound cyanide, thereby contributing to the stabilization of the horseradish peroxidase-cyanide complex and suggesting that the distal arginine will be able to contribute with a similar interaction during stabilization of a bound peroxy transition state and subsequent O-O bond cleavage. The catalytic arginine is additionally engaged in an extensive hydrogen bonding network, which also includes the catalytic distal histidine, a water molecule and Pro(139), a proline residue conserved within the plant peroxidase superfamily. Based on the observed hydrogen bonding network and previous spectroscopic and kinetic work, a general mechanism of peroxidase substrate oxidation is proposed.

Binding Sites↗
PubMed1998

Structural interactions between horseradish peroxidase C and the substrate benzhydroxamic acid determined by X-ray crystallography.

The three-dimensional structure of recombinant horseradish peroxidase in complex with BHA (benzhydroxamic acid) is the first structure of a peroxidase-substrate complex demonstrating the existence of an aromatic binding pocket. The crystal structure of the peroxidase-substrate complex has been determined to 2.0 A resolution with a crystallographic R-factor of 0.176 (R-free = 0. 192). A well-defined electron density for BHA is observed in the peroxidase active site, with a hydrophobic pocket surrounding the aromatic ring of the substrate. The hydrophobic pocket is provided by residues H42, F68, G69, A140, P141, and F179 and heme C18, C18-methyl, and C20, with the shortest distance (3.7 A) found between heme C18-methyl and BHA C63. Very little structural rearrangement is seen in the heme crevice in response to substrate binding. F68 moves to form a lid on the hydrophobic pocket, and the distal water molecule moves 0.6 A toward the heme iron. The bound BHA molecule forms an extensive hydrogen bonding network with H42, R38, P139, and the distal water molecule 2.6 A above the heme iron. This remarkably good match in hydrogen bond requirements between the catalytic residues of HRPC and BHA makes the extended interaction between BHA and the distal heme crevice of HRPC possible. Indeed, the ability of BHA to bind to peroxidases, which lack a peripheral hydrophobic pocket, suggests that BHA is a general counterpart for the conserved hydrogen bond donors and acceptors of the distal catalytic site. The closest aromatic residue to BHA is F179, which we predict provides an important hydrophobic interaction with more typical peroxidase substrates.

Binding Sites↗
PubMed1998

Structure of barley grain peroxidase refined at 1.9-A resolution. A plant peroxidase reversibly inactivated at neutral pH.

The crystal structure of the major peroxidase of barley grain (BP 1) has been solved by molecular replacement and phase combination and refined to an R-factor of 19.2% for all data between 38 and 1.9 A. The refined model includes amino acid residues 1-309, one calcium ion, one sodium ion, iron-protoporphyrin IX, and 146 solvent molecules. BP 1 has the apparently unique property of being unable to catalyze the reaction with the primary substrate hydrogen peroxide to form compound I at pH values > 5, a feature investigated by obtaining crystal structure data at pH 5.5, 7.5, and 8.5. Structural comparison shows that the overall fold of inactive barley grain peroxidase at these pH values resembles that of both horseradish peroxidase C and peanut peroxidase. The key differences between the structures of active horseradish peroxidase C and inactive BP 1 include the orientation of the catalytic distal histidine, disruption of a hydrogen bond between this histidine and a conserved asparagine, and apparent substitution of calcium at the distal cation binding site with sodium at pH 7.5. These profound changes are a result of a dramatic structural rearrangement to the loop region between helices B and C. This is the first time that structural rearrangements linked to active site chemistry have been observed by crystallography in the peroxidase domain distal to heme.

Crystallography, X-Ray↗
PubMed1997

ENVIRONMENTAL AUDITING: Exceedance of Critical Loads for Lakes inFinland, Norway, and Sweden: Reduction Requirements for AcidifyingNitrogen and Sulfur Deposition

/ The main objectives of this study were to identify the regions inFennoscandia where the critical loads of sulfur (S) and acidifying nitrogen(N) for lakes are exceeded and to investigate the consequences for depositionreductions, with special emphasis on the possible trade-offs between S and Ndeposition in order to achieve nonexceedance. In the steady-state model forcalculating critical loads and their exceedances, all relevant processesacting assinks for N and S are considered. The critical loads of N and S areinterrelated (defining the so-called critical load function), and therefore asingle critical load for one pollutant cannot be defined without makingassumptions about the other. Comparing the present N and S deposition withthe critical load function for each lake allows determination of thepercentage of lakes in the different regions of Fennoscandia where: (1) Sreductions alone can achieve nonexceedance, (2) N reductions alone aresufficient, and (3) both N and S reductions are required but to a certaindegree interchangeable. Secondly, deposition reduction requirements wereassessed by fixing the N deposition to the present level, in this wayanalyzing the reductions required for S, and by computing the percentage oflakes exceeded in Finland, Norway and Sweden for every possible percentdeposition reduction in S and N, in this way showing the (relative)effectiveness of reducing S and/or N deposition. The results showed clearregional patterns in the S and N reduction requirements. In practically thewhole of Finland and the northern parts of Scandinavia man-made acidificationof surface waters could be avoided by reducing S deposition alone. In thesouthern parts of Sweden some reductions in N deposition are clearly neededin addition to those for S. In southern Norway strong reductions are requiredfor both N and S deposition.KEY WORDS: Acidification; Critical load; Exceedance; Sulfur; Nitrogen;Deposition; Lake

Journal Article↗
PubMed1997

Crystal structure of horseradish peroxidase C at 2.15 A resolution.

The crystal structure of horseradish peroxidase isozyme C (HRPC) has been solved to 2.15 A resolution. An important feature unique to the class III peroxidases is a long insertion, 34 residues in HRPC, between helices F and G. This region, which defines part of the substrate access channel, is not present in the core conserved fold typical of peroxidases from classes I and II. Comparison of HRPC and peanut peroxidase (PNP), the only other class III (higher plant) peroxidase for which an X-ray structure has been completed, reveals that the structure in this region is highly variable even within class III. For peroxidases of the HRPC type, characterized by a larger FG insertion (seven residues relative to PNP) and a shorter F' helix, we have identified the key residue involved in direct interactions with aromatic donor molecules. HRPC is unique in having a ring of three peripheral Phe residues, 142, 68 and 179. These guard the entrance to the exposed haem edge. We predict that this aromatic region is important for the ability of HRPC to bind aromatic substrates.

Amino Acid Sequence↗
PubMed1996

Structure of porcine pancreatic spasmolytic polypeptide at 1.95 A resolution.

The structure of a trigonal crystal form of porcine pancreatic spasmolytic polypeptide (PSP) has been solved by molecular replacement and refined to 1.95 A resolution. Three heavy-atom derivatives were prepared, giving unbiased phase information, which was used in the model building of the protein molecules. The final conventional R value is 19.8% with the inclusion of 183 water molecules. PSP crystallizes as a dimer in space group P3(1)21 with a non-crystallographic twofold axis relating the monomers. The monomer consists of two very similar domains each composed of three loop regions. Two clefts are found in the monomer, one in each domain, that are proposed as possible substrate-binding sites. Important interactions have been identified in the proposed substrate-binding sites, where conserved water molecules probably mimic the hydrophilic positions of the substrate. The estimated cleft size is 9 x 9 x 12 A. Analysis of the charge distribution within the clefts, by an electrostatic potential calculation, shows the clefts to be essentially non-charged.

Journal Article↗
PubMed1996

A flexible loop at the dimer interface is a part of the active site of the adjacent monomer of Escherichia coli orotate phosphoribosyltransferase.

Orotate phosphoribosyltransferase (OPRTase) is involved in the biosynthesis of pyrimidine nucleotides. Alpha-D-ribosyldiphosphate 5-phosphate (PRPP) and orotate are utilized to form pyrophosphate and orotidine 5'-monophosphate (OMP) in the presence of divalent cations, preferably Mg2+. OMP is thereafter converted to uridine 5'-monophosphate by OMP decarboxylase. We have determined the 2.4 angstrom structure of Escherichia coli OPRTase, ligated with sulfate, by molecular replacement and refined the structure to an R-factor of 18.3% for all data. In the structure of the E. coli enzyme we have determined the fold of a flexible loop region with a highly conserved amino acid sequence among OPRTases, a region known to take part in catalysis. The structure of this region was not determined in the model used for molecular replacement, and it involves interactions at the dimer interface through a bound sulfate ion. Crystalline E. coli OPRTase is a homodimer, with sulfate ions inhibiting enzyme activity bound in the dimer interface close to the flexible loop region. Although this loop is very close in space to the sulfate binding site, and sulfate is found in both interfaces of the homodimer, the loop structure is only traceable in one monomer. We expect that the mobility of this loop is important for catalysis, and, on the basis of the reported structure and the structure of Salmonella typhimurium OPRTase.OMP, we propose that the movement of this loop in association with the movement of OMP is vital to catalysis. Apart from the flexible loop region and a solvent-exposed loop (residues 158-164), the most significant differences in structure between S. typhimurium OPRTase.OMP and E. coli OPRTase are found in the substrate binding regions: the 5'-phosphate binding region (residues 120-131), the binding region for the orotate part of OMP (residues 25-27), and the pyrophosphate binding region (residues 71-73).

Amino Acid Sequence↗
PubMed1995

Crystallization and preliminary X-ray studies of recombinant horseradish peroxidase.

A non-glycosylated form of horseradish peroxidase c extracted from Escherichia coli inclusion bodies and refolded in the presence of haem and Ca(2+) ions has been used to grow protein crystals suitable for X-ray diffraction analysis. The crystals are prisms in the trigonal space group P3(1)12 or P3(2)12 with a = b = 158.9 and c = 114.3 A, and diffract to 1.9 A. There are four molecules, each of 34 kDa, in the asymmetric unit. The molecules of the asymmetric unit are related by approximate translational symmetry, resulting in pseudo-centerings. Data to approximately 15 A can thus be described by a lattice of a' = b' = 91.7 A and c' = 57.1 A, alpha = beta = 90 degrees and gamma = 120 degrees, including four molecules.

Journal Article↗