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K F Jensen

Publications and source records attributed to K F Jensen.

At least 19 recordsLinked to original sources
PubMed2000

Structural basis for the catalytic mechanism of a proficient enzyme: orotidine 5'-monophosphate decarboxylase.

Orotidine 5'-monophosphate decarboxylase (ODCase) catalyzes the decarboxylation of orotidine 5'-monophosphate, the last step in the de novo synthesis of uridine 5'-monophosphate. ODCase is a very proficient enzyme [Radzicka, A., and Wolfenden, R. (1995) Science 267, 90-93], enhancing the reaction rate by a factor of 10(17). This proficiency has been enigmatic, since it is achieved without metal ions or cofactors. Here we present a 2.5 A resolution structure of ODCase complexed with the inhibitor 1-(5'-phospho-beta-D-ribofuranosyl)barbituric acid. It shows a closely packed dimer composed of two alpha/beta-barrels with two shared active sites. The orientation of the orotate moiety of the substrate is unambiguously deduced from the structure, and previously proposed catalytic mechanisms involving protonation of O2 or O4 can be ruled out. The proximity of the OMP carboxylate group with Asp71 appears to be instrumental for the decarboxylation of OMP, either through charge repulsion or through the formation of a very short O.H.O hydrogen bond between the two carboxylate groups.

Amino Acid Sequence↗
PubMed2000

Neurotoxicity of ethyl methacrylate in rats.

Ethyl methacrylate (ethyl 2-methyl-2-propenoate, EMA) has been implicated in the development of neurologic impairment following occupational exposure. The potential of EMA to produce neurotoxicity was investigated in adult male Sprague-Dawley rats in two experiments. In the first experiment, animals were administered 100, 200, 400, or 800 mg/kg by daily intraperitoneal (i.p.) injections for 60 d. Control rats received daily i.p. injections of 1 ml saline/kg. Clinical observations, spontaneous motor activity, and performance in the Morris water maze were assessed. Alterations in clinical parameters in the higher dose groups included lethargy, impaired breathing, decreased weight gain, and increased mortality. Alterations in motor activity were observed at 100 mg/kg, a dose that did not cause alterations in clinical parameters, body weight gain, or mortality. There was also a dose-dependent impairment in performance in the Morris water maze. In the second experiment, animals were administered EMA in drinking water at concentrations of 0.1, 0.2, or 0.5% for 60 d. Control rats were administered tap water. Animals were perfused at the termination of exposure and samples of brain, spinal cord, and sciatic nerve were prepared for histological examination. Spongiform alterations were observed in fiber tracts of the forebrain, brainstem, and spinal cord. Clusters of axonal swellings were scattered throughout the dorsal, ventral, and lateral columns of the spinal cord, and typically involved internodal segments of two or three neighboring axons. Shrunken axons with separated myelin lamellae and large axons with thinner than normal myelin sheaths were apparent in the sciatic nerve. The patterns of alterations in the white matter of the spinal cord and the sciatic nerve are consistent with myelinopathy, but additional experiments are necessary to confirm whether oligodendroglia and Schwann cells are the primary sites of injury. In addition to the alterations associated with myelin, there was a decrease in the density of neurons in the ventral horn of the spinal cord. While the observed effects of EMA on the nervous system of rats are consistent with neurologic symptoms of workers exposed to EMA, additional experiments are necessary to determine if the level and route of exposures associated with occupational use produce these impairments in experimental animals.

Administration, Oral↗
PubMed2000

Crystallization and preliminary X-ray studies of membrane-associated Escherichia coli dihydroorotate dehydrogenase.

Dihydroorotate dehydrogenases (DHODs) are flavin-containing enzymes which catalyse the conversion of (S)-dihydroorotate to orotate, the fourth step in the de novo biosynthesis of pyrimidine nucleotides. Two major families of DHODs have now been identified based on their amino-acid sequence similarities. The two families differ in their reaction mechanisms, but structures are only known of enzymes belonging to family 1. DHOD from Escherichia coli is a typical member of family 2, which contains the membrane-associated enzymes from Gram-negative bacteria and eukaryotes. Yellow crystals grown of this enzyme belong to the space group P4(1)2(1)2 or P4(3)2(1)2. The unit-cell parameters are a = b = 119.2, c = 294.3 A. Owing to the rather large c axis, the currently available resolution of data is 2.2 A.

Cell Membrane↗
PubMed1999

Kinetic mechanism of uracil phosphoribosyltransferase from Escherichia coli and catalytic importance of the conserved proline in the PRPP binding site.

Phosphoribosyltransferases catalyze the formation of nucleotides from a nitrogenous base and 5-phosphoribosyl-alpha-1-pyrophosphate (PRPP). These enzymes and the PRPP synthases resemble each other in a short homologous sequence of 13 amino acid residues which has been termed the PRPP binding site and which interacts with the ribose 5-phosphate moiety in structurally characterized complexes of PRPP and nucleotides. We show that each class of phosphoribosyltransferases has subtle deviations from the general consensus PRPP binding site and that all uracil phosphoribosyltransferases (UPRTases) have a proline residue at a position where other phosphoribosyltransferases and the PRPP synthases have aspartate. To investigate the role of this unusual proline (Pro 131 in the E. coli UPRTase) for enzyme activity, we changed the residue to an aspartate and purified the mutant P131D enzyme to compare its catalytic properties with the properties of the wild-type protein. We found that UPRTase of E. coli obeyed the kinetics of a sequential mechanism with the binding of PRPP preceding the binding of uracil. The basic kinetic constants were derived from initial velocity measurements, product inhibition, and ligand binding assays. The change of Pro 131 to Asp caused a 50-60-fold reduction of the catalytic rate (kcat) in both directions of the reaction and approximately a 100-fold increase in the KM for uracil. The KM for PRPP was strongly diminished by the mutation, but kcat/KM,PRPP and the dissociation constant (KD,PRPP) were nearly unaffected. We conclude that the proline in the PRPP binding site of UPRTase is of only little importance for binding of PRPP to the free enzyme, but is critical for binding of uracil to the enzyme-PRPP complex and for the catalytic rate.

Amino Acid Sequence↗
PubMed1999

The activity of Escherichia coli dihydroorotate dehydrogenase is dependent on a conserved loop identified by sequence homology, mutagenesis, and limited proteolysis.

Dihydroorotate dehydrogenase catalyzes the oxidation of dihydroorotate to orotate. The enzyme from Escherichia coli was overproduced and characterized in comparison with the dimeric Lactococcus lactis A enzyme, whose structure is known. The two enzymes represent two distinct evolutionary families of dihydroorotate dehydrogenases, but sedimentation in sucrose gradients suggests a dimeric structure also of the E. coli enzyme. Product inhibition showed that the E. coli enzyme, in contrast to the L. lactis enzyme, has separate binding sites for dihydroorotate and the electron acceptor. Trypsin readily cleaved the E. coli enzyme into two fragments of 182 and 154 residues, respectively. Cleavage reduced the activity more than 100-fold but left other molecular properties, including the heat stability, intact. The trypsin cleavage site, at R182, is positioned in a conserved region that, in the L. lactis enzyme, forms a loop where a cysteine residue is very critical for activity. In the corresponding position, the enzyme from E. coli has a serine residue. Mutagenesis of this residue (S175) to alanine or cysteine reduced the activities 10000- and 500-fold, respectively. The S175C mutant was also defective with respect to substrate and product binding. Structural and mechanistic differences between the two different families of dihydroorotate dehydrogenase are discussed.

Binding Sites↗
PubMed1999

Kinetic mechanism of OMP synthase: a slow physical step following group transfer limits catalytic rate.

Orotate phosphoribosyltransferase (OMP synthase, EC 2.4.2.10) forms the UMP precursor orotidine 5'-monophophate (OMP) from orotate and alpha-D-5-phosphoribosyl-1-pyrophosphate (PRPP). Here, equilibrium binding, isotope partitioning, and chemical quench studies were used to determine rate and equilibrium constants for the kinetic mechanism. PRPP bound to two sites per dimer with a KD of 33 microM. Binding of OMP and orotate also occurred to a single class of two sites per dimer, with KD values of 3 and 280 microM, respectively. Pyrophosphate binding to two sites was weak with a KD of 960 microM, and in the presence of bound orotate, its affinity for the first site was enhanced 4-fold (KD = 230 microM). Preformed E.OMP, E.PRPP, E.PPi, and E.orotate complexes were trapped as products in isotope partitioning experiments, indicating that each was catalytically competent and confirming a random mechanism. Rapid quench experiments revealed burst kinetics for product formation in both the forward phosphoribosyltransferase and the reverse pyrophosphorolysis reactions. The steady-state rate in the forward reaction was preceded by a burst (nfwd = 1.5/dimer) of at least 300 s-1. In the pyrophosphorolysis reaction, a burst (nrev = 0.7/dimer; k >/= 300 s-1) was also noted. These results allowed us to develop a complete kinetic mechanism for OPRTase, in which a rapid phosphoribosyl transfer reaction at equilibrium is followed by a slow step involving release of product. When the microviscosity, etarel, of the reaction medium was increased with sucrose, the forward kcat decreased in proportion to etarel with a slope of 0.8. In the reverse reaction a more limited dependence of kcat (slope = 0. 3) was observed. On the basis of the known structures of OPRTase, we propose that a highly conserved, catalytically important, solvent-exposed loop descends during catalysis to shield the active site. In the accompanying paper, the slow product release step is shown to relate to movement of the solvent-exposed loop.

Binding Sites↗
PubMed1998

Chronic administration of aluminum-fluoride or sodium-fluoride to rats in drinking water: alterations in neuronal and cerebrovascular integrity.

This study describes alterations in the nervous system resulting from chronic administration of the fluoroaluminum complex (AlF3) or equivalent levels of fluoride (F) in the form of sodium-fluoride (NaF). Twenty seven adult male Long-Evans rats were administered one of three treatments for 52 weeks: the control group was administered double distilled deionized drinking water (ddw). The aluminum-treated group received ddw with 0.5 ppm AlF3 and the NaF group received ddw with 2.1 ppm NaF containing the equivalent amount of F as in the AlF3 ddw. Tissue aluminum (Al) levels of brain, liver and kidney were assessed with the Direct Current Plasma (DCP) technique and its distribution assessed with Morin histochemistry. Histological sections of brain were stained with hematoxylin & eosin (H&E), Cresyl violet, Bielschowsky silver stain, or immunohistochemically for beta-amyloid, amyloid A, and IgM. No differences were found between the body weights of rats in the different treatment groups although more rats died in the AlF3 group than in the control group. The Al levels in samples of brain and kidney were higher in both the AlF3 and NaF groups relative to controls. The effects of the two treatments on cerebrovascular and neuronal integrity were qualitatively and quantitatively different. These alterations were greater in animals in the AlF3 group than in the NaF group and greater in the NaF group than in controls.

Aluminum Compounds↗
PubMed1998

The crystal structure of Lactococcus lactis dihydroorotate dehydrogenase A complexed with the enzyme reaction product throws light on its enzymatic function.

Dihydroorotate dehydrogenases (DHODs) catalyze the oxidation of (S)-dihydroorotate to orotate, the fourth step and only redox reaction in the de novo biosynthesis of pyrimidine nucleotides. A description is given of the crystal structure of Lactococcus lactis dihydroorotate dehydrogenase A (DHODA) complexed with the product of the enzyme reaction orotate. The structure of the complex to 2.0 A resolution has been compared with the structure of the native enzyme. The active site of DHODA is known to contain a water filled cavity buried beneath a highly conserved and flexible loop. In the complex the orotate displaces the water molecules from the active site and stacks above the DHODA flavin isoalloxazine ring, causing only small movements of the surrounding protein residues. The orotate is completely buried beneath the protein surface, and the orotate binding causes a significant reduction in the mobility of the active site loop. The orotate is bound by four conserved asparagine side chains (Asn 67, Asn 127, Asn 132, and Asn 193), the side chains of Lys 43 and Ser 194, and the main chain NH groups of Met 69, Gly 70, and Leu 71. Of these the Lys 43 side chain makes hydrogen bonds to both the flavin isoalloxazine ring and the carboxylate group of the orotate. Potential interactions with bound dihydroorotate are considered using the orotate complex as a basis for molecular modeling. The role of Cys 130 as the active site base is discussed, and the sequence conservation of the active site residues across the different families of DHODs is reviewed, along with implications for differences in substrate binding and in the catalytic mechanisms between these families.

Binding Sites↗
PubMed1997

Active site of dihydroorotate dehydrogenase A from Lactococcus lactis investigated by chemical modification and mutagenesis.

The flavin-containing enzyme dihydroorotate dehydrogenase (DHOD) catalyzes the oxidation of dihydroorotate (DHO) to orotate, the first aromatic intermediate in pyrimidine biosynthesis. The first structure of a DHOD, the A form of the enzyme from Lactococcus lactis, has recently become known, and some conserved residues were suggested to have a role in the active site [Rowland et al. (1997) Structure 2, 239-252]. In particular, Cys 130 was hypothesized to work as a base, which activates dihydroorotate (DHO) for hydride transfer. By chemical modification and site-directed mutagenesis we have obtained results consistent with this proposal. Cys 130 was susceptible to alkylating reagents, and mutants of Cys 130 (C130A and C130S) showed hardly detectable enzyme activity at pH 8.0, while at pH 10 the C130S mutant enzyme had approximately 1% of wild-type activity. Mutants of Lys 43, Asn 132, and Lys 164 were also constructed. Exchange of Lys 43 to Ala or Glu (K43A and K43E) and of Asn 132 to Ala (N132A) affected both catalysis and substrate binding. Expressed as kcat/KM for DHO, the deterioration of these three mutant enzymes was 10(3)-10(4)-fold. Flavin spectra of the mutant enzymes were not, like the wild-type enzyme, bleached by DHO in stopped-flow experiments, showing that they were deficient with respect to the first half-reaction, namely reduction of FMN by DHO, which was not rate limiting for the wild-type enzyme. The binding interaction between flavin and the reaction product, orotate, could be monitored by a red shift of the flavin absorbance in the wild-type enzyme. The C130A, C130S, and N132A mutant enzymes displayed similar capacity to bind orotate. In contrast, orotate did not change the absorption spectra of the K43 mutant enzymes, although it did inhibit their activity. All of the mutant enzymes, except K164A, contained normal levels of flavin. The results are discussed in relation to the structures of DHODA and other flavoenzymes. The possible acid-base chemistry of Cys 130 is compared to previous work on mammalian dihydropyrimidine dehydrogenases, flavoenzymes, which catalyze the reversed reaction, namely the reduction of pyrimidine bases.

Alkylating Agents↗
PubMed1997

NusA is required for ribosomal antitermination and for modulation of the transcription elongation rate of both antiterminated RNA and mRNA.

Ribosomal RNA (rRNA) is elongated twice as fast as mRNA in vivo due to the presence of antitermination sequences in the 5' part of the rRNA transcripts. A number of Nus factors bind to RNA polymerase at the antitermination sites and help confer resistance to Rho-dependent termination of transcription. In this paper, the effects of the nusAcs10 allele on the elongation rate of both mRNA and antiterminated RNA were investigated. The results indicate that NusA is required to achieve a high elongation rate of RNA chains carrying the ribosomal antitermination boxA and that antitermination is defective when the rate of transcription elongation is decreased by the nusAcs10 allele. Furthermore, the nusAcs10 allele had no significant effects on the elongation rate of normal lacZ mRNA during steady state growth, but it abolished the inhibition of lacZ mRNA elongation by guanosine 3',5'-bis(diphosphate) (ppGpp). These results suggest that NusA is the component of the transcription elongation complex required for inhibition of mRNA elongation by ppGpp.

Alleles↗
PubMed1997

The crystal structure of the flavin containing enzyme dihydroorotate dehydrogenase A from Lactococcus lactis.

BACKGROUND: . Dihydroorotate dehydrogenase (DHOD) is a flavin mononucleotide containing enzyme, which catalyzes the oxidation of (S)-dihydroorotate to orotate, the fourth step in the de novo biosynthesis of pyrimidine nucleotides. Lactococcus lactis contains two genes encoding different functional DHODs whose sequences are only 30% identical. One of these enzymes, DHODA, is a highly efficient dimer, while the other, DHODB, shows optimal activity only in the presence of an iron-sulphur cluster containing protein with which it forms a complex tetramer. Sequence alignments have identified three different families among the DHODs: the two L. lactis enzymes belong to two of the families, whereas the enzyme from E. coli is a representative of the third. As no three-dimensional structures of DHODs are currently available, we set out to determine the crystal structure of DHODA from L. lactis. The differences between the two L. lactis enzymes make them particularly interesting for studying flavoprotein redox reactions and for identifying the differences between the enzyme families. RESULTS: . The crystal structure of DHODA has been determined to 2.0 resolution. The enzyme is a dimer of two crystallographically independent molecules related by a non-crystallographic twofold axis. The protein folds into and alpha/beta barrel with the flavin molecule sitting between the top of the barrel and a subdomain formed by several barrel inserts. Above the flavin isoalloxazine ring there is a small water filled cavity, completely buried beneath the protein surface and surrounded by many conserved residues. This cavity is proposed as the substrate-binding site. CONCLUSIONS: . The crystal structure has allowed the function of many of the conserved residues in DHODs to be identified: many of these are associated with binding the flavin group. Important differences were identified in some of the active-site residues which vary across the distinct DHOD families, implying significant mechanistic differences. The substrate cavity, although buried, is located beneath a highly conserved loop which is much less ordered than the rest of the protein and may be important in giving access to the cavity. The location of the conserved residues surrounding this cavity suggests the potential orientation of the substrate.

Amino Acid Sequence↗
PubMed1996

The B form of dihydroorotate dehydrogenase from Lactococcus lactis consists of two different subunits, encoded by the pyrDb and pyrK genes, and contains FMN, FAD, and [FeS] redox centers.

The B form of dihydroorotate dehydrogenase from Lactococcus lactis (DHOdehase B) is encoded by the pyrDb gene. However, recent genetic evidence has revealed that a co-transcribed gene, pyrK, is needed to achieve the proper physiological function of the enzyme. We have purified DHOdehase B from two strains of Escherichia coli, which harbored either the pyrDb gene or both the pyrDb and the pyrK genes of L. lactis on multicopy plasmids. The enzyme encoded by pyrDb alone (herein called the delta-enzyme) was a bright yellow, dimeric protein that contained one molecule of tightly bound FMN per subunit. The delta-enzyme exhibited dihydroorotate dehydrogenase activity with dichloroindophenol, potassium hexacyanoferrate(III), and molecular oxygen as electron acceptors but could not use NAD+. The DHOdehase B purified from the E. coli strain that carried both the pyrDb and pyrK genes on a multicopy plasmid (herein called the deltakappa-enzyme) was quite different, since it was formed as a complex of equal amounts of the two polypeptides, i.e. two PyrDB and two PyrK subunits. The deltakappa-enzyme was orange-brown and contained 2 mol of FAD, 2 mol of FMN, and 2 mol of [2Fe-2S] redox clusters per mol of native protein as tightly bound prosthetic groups. The deltakappa-enzyme was able to use NAD+ as well as dichloroindophenol, potassium hexacyanoferrate(III), and to some extent molecular oxygen as electron acceptors for the conversion of dihydroorotate to orotate, and it was a considerably more efficient catalyst than the purified delta-enzyme. Based on these results and on analysis of published sequences, we propose that the architecture of the deltakappa-enzyme is representative for the dihydroorotate dehydrogenases from Gram-positive bacteria.

Amino Acid Sequence↗
PubMed1996

Different oligomeric states are involved in the allosteric behavior of uracil phosphoribosyltransferase from Escherichia coli.

Uracil phosphoribosyltransferase, catalyzing the formation of UMP and pyrophosphate from uracil and 5-phosphoribosyl-alpha-1-diphosphate (PPRibP), was purified from an overproducing strain of Escherichia coli. GTP was shown to activate the enzyme by reducing K(m) for PPRibP by about fivefold without affecting Vmax. When started by addition of enzyme, the reactions accelerated over an extended period of time, while enzyme solutions incubated first with GTP and PPRibP displayed constant velocities. This indicated that PPRibP and GTP influenced the structure of the enzyme. Gel-filtration and sedimentation analyses showed that the apparent oligomeric state of uracil phosphoribosyltransferase is defined by a dynamic equilibrium between a slowly sedimenting form (dimeric or trimeric) that has only a little activity, and a more highly aggregated form (pentameric or hexameric), which is more active. It appears that the smaller form predominates in the absence of substrates, while the larger form predominates in the presence of GTP and PPRibP. Guanosine-3',5'-bis(diphosphate) was found to activate the enzyme much like GTP.

Allosteric Regulation↗
PubMed1996

Uracil phosphoribosyltransferase from the extreme thermoacidophilic archaebacterium Sulfolobus shibatae is an allosteric enzyme, activated by GTP and inhibited by CTP.

Uracil phosphoribosyltransferase, which catalyses the formation of UMP and pyrophosphate from uracil and 5-phosphoribosyl alpha-1-pyrophosphate (PRPP), was partly purified from the extreme thermophilic archaebacterium Sulfolobus shibatae. The enzyme required divalent metal ions for activity and it showed the highest activity at pH 6.4. The specific activity of the enzyme was 50-times higher at 95 degrees C than at 37 degrees C, but the functional half-life was short at 95 degrees C. The activity of uracil phosphoribosyltransferase was strongly activated by GTP, which increased Vmax of the reaction by approximately 20-fold without much effect on K(m) for the substrates. The concentration of GTP required for half-maximal activation was about 80 microM. CTP was a strong inhibitor and acted by raising the concentration of GTP needed for half-maximal activation of the enzyme. We conclude that uracil phosphoribosyltransferase from S. shibatae is an allosteric enzyme which is activated by a purine nucleotide and inhibited by a pyrimidine nucleotide as seen for several enzymes in the pyrimidine nucleotide biosynthetic pathway of Escherichia coli, but not observed before for any phosphoribosyltransferase.

Adenosine Triphosphate↗
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↗
PubMed1996

Purification and characterization of dihydroorotate dehydrogenase A from Lactococcus lactis, crystallization and preliminary X-ray diffraction studies of the enzyme.

Lactococcus lactis is the only organism known to contain two dihydroorotate dehydrogenases, i.e., the A- and B-forms. In this paper, we report the overproduction, purification, and crystallization of dihydroorotate dehydrogenase A. In solution, the enzyme is bright yellow. It is a dimer of subunits (34 kDa) that contain one molecule of flavin mononucleotide each. The enzyme shows optimal function in the pH range 7.5-9.0. It is specific for L-dihydroorotate as substrate and can use dichlorophenolindophenol, potassium hexacyanoferrate (III), and, to a lower extent, also molecular oxygen as acceptors of the reducing equivalents, whereas the pyridine nucleotide coenzymes (NAD+, NADP+) and the respiratory quinones (i.e., vitamins Q6, Q10 and K2) were inactive. The enzyme has been crystallized from solutions of 30% polyethylene glycol, 0.2 M sodium acetate, and 0.1 M Tris-HCl, pH 8.5. The resulting yellow crystals diffracted well and showed little sign of radiation damage during diffraction experiments. The crystals are monoclinic, space group P21 with unit cell dimensions a = 54.19 A, b = 109.23 A, c = 67.17 A, and beta = 104.5 degrees. A native data set has been collected with a completeness of 99.3% to 2.0 A and an Rsym value of 5.2%. Analysis of the solvent content and the self-rotation function indicates that the two subunits in the asymmetric unit are related by a noncrystallographic twofold axis perpendicular to the crystallographic b and c axes.

Bacterial Proteins↗
PubMed1996

Increased neurotoxicity following concurrent exposure to pyridostigmine bromide, DEET, and chlorpyrifos.

The operating environment of the service personnel during the Persian Gulf War involved psychological, biological, and chemical elements including exposure to pesticides such as the insect repellent DEET (N,N-diethyl-m-toluamide) and the insecticide chlorpyrifos (O,O-diethyl O-3,5,6-trichloropyridinyl phosphorothioate) and to pyridostigmine bromide (PB,3-dimethylaminocarbonyloxy-N-methylpyridinium bromide) that was administered as a prophylactic agent against possible nerve gas attack. The present study was designed to determine the toxicity produced by individual or coexposure of hens 5 days/week for 2 months to 5 mg PB/kg/day in water, by gavage; 500 mg DEET/kg/day, neat, sc; and 10 mg chlorpyrifos kg/day in corn oil, sc. Coexposure to various binary treatments produced greater neurotoxicity than that caused by individual exposures and was characterized by severe neurologic deficit and neuropathological alterations. Also, neurotoxicity was further enhanced following concurrent administration of the three chemicals. Severe inhibition of plasma butyrylcholinesterase (BuChE) activity was produced in hens treated with PB (activity 17% of control) compared to those treated with chlorpyrifos (activity 51% of control) or DEET (activity 83% of control). BuChE inhibition was further increased in binary and tertiary treatment groups compared to individual treatment groups. In contrast, a significant inhibition of brain acetylcholinesterase (AChE) was produced in hens administered chlorpyrifos alone (activity 67% of control), while those given chlorpyrifos in combination with other compounds exhibited a significant inhibition of brain AChE activity ranging from 43 to 76%. Brain neurotoxicity target esterase (NTE) was not inhibited in any of the individual treatment groups or PB/DEET, but was significantly inhibited and had activity expressed as a percentage of control in groups administered combined chlorpyrifos with PB of 73% or DEET of 74% and in the tertiary treatment group of 71%. We hypothesize that test compounds may compete for xenobiotic metabolizing enzymes in the liver and blood and may also compromise the integrity of the blood-brain barrier, leading to an increase in their "effective concentrations" in the nervous system to levels equivalent to the toxic doses of individual compounds. This is consistent with the present observation of increases in (1) the inhibition of brain AChE and NTE, (2) the extent of neurologic dysfunction, and (3) the severity and frequency of neuropathologic lesions in the combined treatment groups compared to those administered individual compounds.

Animals↗