Structural characterizations of glycerol kinase: unraveling phosphorylation-induced long-range activation.

Glycerol metabolism provides a central link between sugar and fatty acid catabolism. In most bacteria, glycerol kinase plays a crucial role in regulating channel/facilitator-dependent uptake of glycerol into the cell. In the firmicute Enterococcus casseliflavus, this enzyme's activity is enhanced by phosphorylation of the histidine residue (His232) located in its activation loop, approximately 25 A from its catalytic cleft. We reported earlier that some mutations of His232 altered enzyme activities; we present here the crystal structures of these mutant GlpK enzymes. The structure of a mutant enzyme with enhanced enzymatic activity, His232Arg, reveals that residues at the catalytic cleft are more optimally aligned to bind ATP and mediate phosphoryl transfer. Specifically, the position of Arg18 in His232Arg shifts by approximately 1 A when compared to its position in wild-type (WT), His232Ala, and His232Glu enzymes. This new conformation of Arg18 is more optimally positioned at the presumed gamma-phosphate location of ATP, close to the glycerol substrate. In addition to structural changes exhibited at the active site, the conformational stability of the activation loop is decreased, as reflected by an approximately 35% increase in B factors ("thermal factors") in a mutant enzyme displaying diminished activity, His232Glu. Correlating conformational changes to alteration of enzymatic activities in the mutant enzymes identifies distinct localized regions that can have profound effects on intramolecular signal transduction. Alterations in pairwise interactions across the dimer interface can communicate phosphorylation states over 25 A from the activation loop to the catalytic cleft, positioning Arg18 to form favorable interactions at the beta,gamma-bridging position with ATP. This would offset loss of the hydrogen bonds at the gamma-phosphate of ATP during phosphoryl transfer to glycerol, suggesting that appropriate alignment of the second substrate of glycerol kinase, the ATP molecule, may largely determine the rate of glycerol 3-phosphate production.

Purification, crystallization and preliminary crystallographic study of a recombinant plant aminoaldehyde dehydrogenase from Pisum sativum.

Aminoaldehydes are products of polyamine degradation and are known to be reactive metabolites that are toxic to living cells at high concentrations. These compounds are catabolized by aminoaldehyde dehydrogenases, which are enzymes that contain a nicotinamide adenine dinucleotide coenzyme. Aminoaldehyde dehydrogenase from Pisum sativum was overexpressed in Escherichia coli, purified and crystallized using the hanging-drop method. A complete data set was collected to 2.8 A resolution at 100 K. Crystals belong to the monoclinic space group P2(1), with unit-cell parameters a = 86.4, b = 216.6, c = 205.4 A, beta = 98.1 degrees. Molecular replacement was performed and led to the identification of six dimers per asymmetric unit.

Evaluation of oxaliplatin exposure of healthcare workers during heated intraperitoneal perioperative chemotherapy (HIPEC).

The aim of this study was to evaluate air and surface contaminations, and internal contamination of healthcare workers during open-abdomen HIPEC using oxaliplatin. Platinum (Pt) was measured in urine of exposed workers and in multiple air and surface samples. Three successive HIPEC procedures were investigated in each of the two hospitals participating in the study. Analysis of air samples did not detect any oxaliplatin contamination. Heavy contamination of the operating table, the floor at the surgeon's feet, and the surgeon's overshoes were observed. Hand contamination was observed in surgeons using double gloves for intra-abdominal chemotherapy administration, but not in those using three sets of gloves. Pt was not detected in urine samples obtained after HIPEC (<5 ng/L). The main risk of HIPEC is related to direct or indirect skin exposure and can be prevented by correct use of adapted protective equipment.

Structural and functional characterization of Escherichia coli UMP kinase in complex with its allosteric regulator GTP.

Bacterial UMP kinases are essential enzymes involved in the multistep synthesis of UTP. They are hexamers regulated by GTP (allosteric activator) and UTP (inhibitor). We describe here the 2.8 angstroms crystal structure of Escherichia coli UMP kinase bound to GTP. The GTP-binding site, situated at 15 angstroms from the UMP-binding site and at 24 angstroms from the ATP-binding site, is delineated by two contiguous dimers. The overall structure, as compared with those bound to UMP, UDP, or UTP, shows a rearrangement of its quaternary structure: GTP induces an 11 degrees opening of the UMP kinase dimer, resulting in a tighter dimer-dimer interaction. A nucleotide-free UMP kinase dimer has an intermediate opening. Superposition of our structure with that of archaeal UMP kinases, which are also hexamers, shows that a loop appears to hamper any GTP binding in archeal enzymes. This would explain the absence of activating effect of GTP on this group of UMP kinases. Among GTP-binding residues, the Asp-93 is the most conserved in bacterial UMP kinases. In the previously published structures of E. coli UMP kinase, this residue was shown to be involved in hydrogen bonds between the subunits of a dimer. Its substitution by an alanine decreases the cooperativity for UTP binding and suppresses the reversal by GTP of UTP inhibition. This demonstrates that the previously described mutual exclusion of these two nucleotides is mediated by Asp-93.

Mechanism-based inhibitors of cytokinin oxidase/dehydrogenase attack FAD cofactor.

Cytokinin oxidases/dehydrogenases (CKOs) mediate catabolic regulation of cytokinin levels in plants. Several substrate analogs containing an unsaturated side chain were studied for their possible inhibitory effect on maize CKO (ZmCKO1) by use of various bioanalytical methods. Two allenic derivatives, N(6)-(buta-2,3-dienyl)adenine (HA-8) and N(6)-(penta-2,3-dienyl)adenine (HA-1), were identified as strong mechanism-based inhibitors of the enzyme. Despite exhaustive dialysis, the enzyme remained inhibited. Conversely, substrate analogs with a triple bond in the side chain were much weaker inactivators. The crystal structures of recombinant ZmCKO1 complexed with HA-1 or HA-8 were solved to 1.95 A resolution. Together with Raman spectra of the inactivated enzyme, it was revealed that reactive imine intermediates generated by oxidation of the allenic inhibitors covalently bind to the flavin adenine dinucleotide (FAD) cofactor. The binding occurs at the C4a atom of the isoalloxazine ring of FAD, the planarity of which is consequently disrupted. All the compounds under study were also analyzed for binding to the Arabidopsis cytokinin receptors AHK3 and AHK4 in a bacterial receptor assay and for cytokinin activity in the Amaranthus bioassay. HA-1 and HA-8 were found to be good receptor ligands with a significant cytokinin activity. Nevertheless, due to their ability to inactivate CKO in the desired time intervals or developmental stages, they both represent attractive compounds for physiological studies, as the inhibition mechanism of HA-1 and HA-8 is mainly FAD dependent.

Structure of Escherichia coli UMP kinase differs from that of other nucleoside monophosphate kinases and sheds new light on enzyme regulation.

Bacterial UMP kinases are essential enzymes involved in the multistep synthesis of nucleoside triphosphates. They are hexamers regulated by the allosteric activator GTP and inhibited by UTP. We solved the crystal structure of Escherichia coli UMP kinase bound to the UMP substrate (2.3 A resolution), the UDP product (2.6 A), or UTP (2.45 A). The monomer fold, unrelated to that of other nucleoside monophosphate kinases, belongs to the carbamate kinase-like superfamily. However, the phosphate acceptor binding cleft and subunit assembly are characteristic of UMP kinase. Interactions with UMP explain the high specificity for this natural substrate. UTP, previously described as an allosteric inhibitor, was unexpectedly found in the phosphate acceptor site, suggesting that it acts as a competitive inhibitor. Site-directed mutagenesis of residues Thr-138 and Asn-140, involved in both uracil recognition and active site interaction within the hexamer, decreased the activation by GTP and inhibition by UTP. These experiments suggest a cross-talk mechanism between enzyme subunits involved in cooperative binding at the phosphate acceptor site and in allosteric regulation by GTP. As bacterial UMP kinases have no counterpart in eukaryotes, the information provided here could help the design of new antibiotics.

Crystal structure of a four-copper laccase complexed with an arylamine: insights into substrate recognition and correlation with kinetics.

Laccases are multicopper oxidases that catalyze the oxidation of a wide range of phenols or arylamines, and their use in industrial oxidative processes is increasing. We purified from the white rot fungus Trametes versicolor a laccase that exists as five different isozymes, depending on glycosylation. The 2.4 A resolution structure of the most abundant isozyme of the glycosylated enzyme was solved. The four copper atoms are present, and it is the first crystal structure of a laccase in its active form. The crystallized enzyme binds 2,5-xylidine, which was used as a laccase inducer in the fungus culture. This arylamine is a very weak reducing substrate of the enzyme. The cavity enclosing 2,5-xylidine is rather wide, allowing the accommodation of substrates of various sizes. Several amino acid residues make hydrophobic interactions with the aromatic ring of the ligand. In addition, two charged or polar residues interact with its amino group. The first one is an histidine that also coordinates the copper that functions as the primary electron acceptor. The second is an aspartate conserved among fungal laccases. The purified enzyme can oxidize various hydroxylated compounds of the phenylurea family of herbicides that we synthesized. These phenolic substrates have better affinities at pH 5 than at pH 3, which could be related to the 2,5-xylidine binding by the aspartate. This is the first high-resolution structure of a multicopper oxidase complexed to a reducing substrate. It provides a model for engineering laccases that are either more efficient or with a wider substrate specificity.

Purification and preliminary crystallographic study of Trametes versicolor laccase in its native form.

Laccases are multi-copper oxidases that catalyse the oxidation of a wide range of phenols and their use in industrial oxidative processes is increasing. A laccase has been purified from the fungus Trametes versicolor and crystallized using the hanging-drop method. Crystals belong to the monoclinic space group P2(1), with unit-cell parameters a = 87.7, b = 110.5, c = 123.2 A, beta = 103.4 degrees. A complete data set was collected to 2.4 A resolution on a Cu K(alpha) rotating-anode X-ray source. Molecular replacement was performed and the initial electron-density maps indicate that the four expected Cu atoms are all present.

Purification, crystallization and preliminary X-ray diffraction study of a recombinant cytokinin oxidase from Zea mays.

Cytokinins are hormones that are involved in plant growth and development. They are irreversibly degraded by cytokinin oxidases/dehydrogenases, flavoenzymes which contain a covalently bound flavine adenine dinucleotide (FAD) cofactor. Cytokinin oxidase from Zea mays (ZmCKO1) was overexpressed in the yeast Yarrowia lipolytica, purified (molecular weight 69 kDa) and crystallized using the hanging-drop method. Crystals belong to the monoclinic space group C2, with unit-cell parameters a = 250.6, b = 50.6, c = 51.5 A, beta = 94.1 degrees. A complete data set has been collected at 100 K to 1.95 A resolution on an X-ray synchrotron source.

Asbestos exposure and radiological abnormalities among maintenance and custodian workers in buildings with friable asbestos-containing materials.

OBJECTIVES: Few studies have been carried out to evaluate the respiratory effects of asbestos exposure of custodian and maintenance workers. METHODS: By a multicentre cross-sectional study, 277 custodian and maintenance employees working in buildings with friable asbestos-containing materials and 87 unexposed subjects were studied for radiological abnormalities by use of the International Labour Office (ILO) classification of radiographs of pneumoconiosis, in relation to parameters of asbestos exposure. RESULTS: The cumulative asbestos exposure index was generally low (fewer than 5 fibres/ml x years in 82.3% of exposed workers). On multivariate analysis, pleural thickening was significantly related to latency since onset of exposure to asbestos in exposed workers, after adjustment for age, body mass index and tobacco smoking. CONCLUSIONS: Asbestos exposure of custodian and maintenance employees in buildings with friable asbestos-containing materials might be associated with an excess of pleural thickening on chest X-rays.

Asbestos-related pleural diseases: dimensional criteria are not appropriate to differentiate diffuse pleural thickening from pleural plaques.

BACKGROUND: In the literature, the criteria used to define pleural plaques (PP) and diffuse pleural thickening (DPT) are very heterogeneous and often imprecise. A multicenter restropective study was conducted to assess the relevance of two radiographic definitions of DPT. METHODS: The study population consisted of 287 subjects with asbestos-related pleural thickening. Two definitions were used to characterize DPT on postero-anterior chest radiographs: definition 1: pleural thickening associated with obliteration of the costophrenic angle; definition 2: pleural thickening at least 5 mm wide, extending for more than one quarter of the chest wall. Prevalence of respiratory symptoms and pulmonary function tests were compared in the DPT and PP groups resulting from the two definitions of DPT. RESULTS: According to definition 1, 34 patients (11.8%) were classified in the DPT group. Prevalence of chronic sputum, dyspnea, and chest pain was significantly higher in this group than in the PP group. FEV(1), FVC, and TLC were significantly lower. The differences persisted after adjustment for confounding factors. According to definition 2,102 patients (36.6%) were classified in the DPT group. DPT and PP groups did not differ in terms of prevalence of respiratory symptoms, or pulmonary function tests. Agreement between readers was significantly better when using definition 1. CONCLUSIONS: Obliteration of costophrenic angle is a much more reliable sign than dimensional criteria to characterize DPT.