Targeting the role of a key conserved motif for substrate selection and catalysis by 3-deoxy-D-manno-octulosonate 8-phosphate synthase

3-Deoxy-D-manno-octulosonate 8-phosphate synthase (KDO8PS) catalyzes the reaction between three-carbon phosphoenolpyruvate (PEP) and five-carbon d-arabinose 5-phosphate (A5P), generating KDO8P, a key intermediate in the biosynthetic pathway to 3-deoxy-D-manno-octulosonate, a component of the lipopolysaccharide of the Gram-negative bacterial cell wall. Both metal-dependent and metal-independent forms of KDO8PS have been characterized. KDO8PS is evolutionarily and mechanistically related to the first enzyme of the shikimate pathway, the obligately divalent metal ion-dependent 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS) that couples PEP and four-carbon D-erythrose 4-phosphate (E4P) to give DAH7P. In KDO8PS, an absolutely conserved KANRS motif forms part of the A5P binding site, whereas in DAH7PS, an absolutely conserved KPR(S/T) motif accommodates E4P. Here, we have characterized four mutants of this motif (AANRS, KAARS, KARS, and KPRS) in metal-dependent KDO8PS from Acidithiobacillus ferrooxidans and metal-independent KDO8PS from Neisseria meningitidis to test the roles of the universal Lys and the Ala-Asn portion of the KANRS motif. The X-ray structures, determined for the N. meningitidis KDO8PS mutants, indicated no gross structural penalty resulting from mutation, but the subtle changes observed in the active sites of these mutant proteins correlated with their altered catalytic function. (1) The AANRS mutations destroyed catalytic activity. (2) The KAARS mutations lowered substrate selectivity, as well as activity. (3) Replacing KANRS with KARS or KPRS destroyed KDO8PS activity but did not produce a functional DAH7PS. Thus, Lys is critical to catalysis, and other changes are necessary to switch substrate specificity for both the metal-independent and metal-dependent forms of these enzymes.     

Oxytocin is expressed by both intrinsic sensory and secretomotor neurons in the enteric nervous system of guinea pig

Single- and double-immunostaining techniques were used systematically to study the distribution pattern and neurochemical density of oxytocin-immunoreactive (-ir) neurons in the digestive tract of the guinea pig. Oxytocin immunoreactivity was distributed widely in the guinea pig gastrointestinal tract; 3%, 13%, 17%, 15%, and 10% of ganglion neurons were immunoreactive for oxytocin in the myenteric plexuses of the gastric corpus, jejunum, ileum, proximal colon, and distal colon, respectively, and 36%, 40%, 52%, and 56% of ganglion neurons were immunoreactive for oxytocin in the submucosal plexuses of the jejunum, ileum, proximal colon, and distal colon, respectively. In the myenteric plexus, oxytocin was expressed exclusively in the intrinsic enteric afferent neurons, as identified by calbindin 28 K. In the submucosal plexuses, oxytocin was expressed in non-cholinergic secretomotor neurons, as identified by vasoactive intestinal polypeptide. Oxytocin-ir nerve fibers in the inner circular muscle layer possibly arose from the myenteric oxytocin-ir neurons, and oxytocin-ir nerve fibers in the mucosa possibly arose from both the myenteric and submucosal oxytocin-ir neurons. Thus, oxytocin in the digestive tract might be involved in gastrointestinal tract motility mainly via the regulation of the inner circular muscle and the balance of the absorption and secretion of water and electrolytes.     

Bleeding time in patients with hepatic cirrhosis.

OBJECTIVE--To determine the frequency of an abnormal bleeding time in patients with cirrhosis and to relate this to known factors that affect primary haemostasis and to the severity of liver disease. DESIGN--Prospective clinical and laboratory study in patients admitted for complications or investigations of liver disease. SETTING--Royal Free Hospital hepatobiliary and liver transplantation unit. SUBJECTS--100 Consecutive inpatients aged 17-74 with various forms of cirrhosis, including alcoholic, biliary, autoimmune, viral, and cryptogenic. At least 10 days had elapsed since any episodes of bleeding, resolution of sepsis, or alcohol intake. No patient was taking any drug known to affect primary haemostasis. MAIN OUTCOME MEASURES--Bleeding time as measured with the Simplate double blade template device. A bleeding time longer than 10 minutes was considered abnormal. Other measures were platelet count, prothrombin time, partial thromboplastin time, packed cell volume, and blood urea, serum bilirubin, and serum albumin concentrations, all measured on each subject at the same time by standard laboratory methods. RESULTS--A weak but significant correlation existed between the bleeding time and the platelet count (rs = 0.483; p less than 0.001). There were significantly lower platelet counts, longer prothrombin times, and higher blood urea and serum bilirubin concentrations in the 42 patients with bleeding times of 10 minutes or more compared with the 58 patients with bleeding times less than 10 minutes. Multiple linear regression analysis showed that the bilirubin concentration as well as the platelet count was independently correlated with the bleeding time. The combination of a platelet count greater than 80 x 10(9)/l and a prothrombin time less than 17 seconds (usually taken as safe limits for performing routine liver biopsy) did not predict a normal bleeding time. Ten of 39 patients fulfilling these criteria had a prolonged bleeding time. CONCLUSIONS--Prolonged bleeding time is common in patients with cirrhosis, even in those with prothrombin times and platelet counts within "safe limits" for invasive procedures. The severity of liver disease as assessed by the bilirubin concentration plays an important part in determining the bleeding time in cirrhosis. The bleeding time should be measured when assessing patients for invasive procedures who have a raised bilirubin concentration or poor hepatic function, even if the platelet count and prothrombin time are considered adequate.     

Refinement of human lysozyme at 1.5 Å resolution analysis of non-bonded and hydrogen-bond interactions

The structure of human lysozyme has been crystallographically refined at 1.5 Å resolution by difference map and restrained least-squares procedures to an R factor of 0.187. A comprehensive analysis of the non-bonded and hydrogen-bonded contacts in the lysozyme molecule, which were not restrained, revealed by the refinement has been carried out. The non-bonded CC contacts begin at ~3.45 Å, and the shorter contacts are dominated, as expected, by interactions between trigonal and tetrahedral carbon atoms. The CO contact distances have a “foot” at 3.05 Å. The CN distance plot shows a significant peak at 3.25 Å, which results from close contact between peptide NHs and carbonyl carbons involved in N_iC′_{i ? 2} interactions in α-helices and reverse turns. The distances involving sulphur atoms discriminate SC trigonal interactions at 3.4 to 3.6 Å from SC tetrahedral interactions at 3.7 Å. All these types of non-bonded interactions show minimum distances close to standard van der Waals' separations.Analysis of hydrogen-bond distances has been carried out by using standard geometry to place hydrogen atoms and measuring the XHO distances. On this basis, there are 130 intramolecular hydrogens: 111 NHO bonds, of which 69 are between main-chain atoms, 13 between side-chain atoms and 29 between mainchain and side-chain atoms. If a cluster of four well-defined internal water molecules is included in the protein structure, there is a total of 19 OHO hydrogen bonds. The mean NO, NHO distances and HN?O angles are 2.96 ± 0.17 Å, 2.05 ± 0.18 Å and 18.5 ± 9.6 °, and the mean OO, OHO distances and HÔO angles are 2.83 ± 0.19 Å, 1.98 ± 0.26 Å and 23.8 ± 13.4 °. The distances agree well with standard values, although the hydrogen bonds are consistently more non-linear than in equivalent small molecules. An analysis of the hydrogen-bond angles at the receptor atom indicates that the α-helix, β-sheet and reverse turn have characteristic angular values. A detailed analysis of the regularity of the α-helices and reverse turns shows small but consistent differences between the α-helices in lysozyme and the current standard model, which may now need revision. Of the 21 reverse turns that include a hydrogen bond, the conformations of 19 agree very closely with four of the five standard types. We conclude that the restrained least-squares method of refinement has been validated by these analyses.     

Targeting conserved water molecules: Design of 4-aryl-5-cyanopyrrolo[2,3-d]pyrimidine Hsp90 inhibitors using fragment-based screening and structure-based optimization

Inhibitors of the Hsp90 molecular chaperone are showing promise as anti-cancer agents. Here we describe a series of 4-aryl-5-cyanopyrrolo[2,3-d]pyrimidine ATP competitive Hsp90 inhibitors that were identified following structure-driven optimization of purine hits revealed by NMR based screening of a proprietary fragment library. Ligand-Hsp90 X-ray structures combined with molecular modeling led to the rational displacement of a conserved water molecule leading to enhanced affinity for Hsp90 as measured by fluorescence polarization, isothermal titration calorimetry and surface plasmon resonance assays. This displacement was achieved with a nitrile group, presenting an example of efficient gain in binding affinity with minimal increase in molecular weight. Some compounds in this chemical series inhibit the proliferation of human cancer cell lines in vitro and cause depletion of oncogenic Hsp90 client proteins and concomitant elevation of the co-chaperone Hsp70. In addition, one compound was demonstrated to be orally bioavailable in the mouse. This work demonstrates the power of structure-based design for the rapid evolution of potent Hsp90 inhibitors and the importance of considering conserved water molecules in drug design.     

Recovery of Sugars from Ionic Liquid Biomass Liquor by Solvent Extraction

The dissolution of biomass into ionic liquids (ILs) has been shown to be a promising alternative biomass pretreatment technology, facilitating faster breakdown of cellulose through the disruption of lignin and the decrystallization of cellulose. Both biological and chemical catalysis have been employed to enhance the conversion of IL-treated biomass polysaccharides into monomeric sugars. However, biomass-dissolving ILs, sugar monomers, and smaller carbohydrate oligomers are all soluble in water. This reduces the overall sugar content in the recovered solid biomass and complicates the recovery and recycle of the IL. Near-complete recovery of the IL and the holocellulose is essential for an IL-based pretreatment technology to be economically feasible. To address this, a solvent extraction technique, based on the chemical affinity of boronates such as phenylboronic acid and naphthalene-2-boronic acid for sugars, was applied to the extraction of glucose, xylose, and cellobiose from aqueous mixtures of 1-ethyl-3-methylimidazolium acetate. It was shown that boronate complexes could extract up to 90% of mono- and disaccharides from aqueous IL solutions, 100% IL systems, and hydrolysates of corn stover containing IL. The use of boronate complexes shows significant potential as a way to recover sugars at several stages in ionic liquid biomass pretreatment processes, delivering a concentrated solution of fermentable sugars, minimizing toxic byproducts, and facilitating ionic liquid cleanup and recycle.     

Chromatographic resolution of nucleic acids extracted from Penicillium chrysogenum

Summary High molecular weight DNA extracted from Penicillium chrysogenum has been fractionated using RPC-5 Analog, into three distinct types designated 1, 2 and 3. Types 1 and 2 have the same buoyant density of 1.710 g/cm³ and together appear to comprise the nuclear DNA. Type 1 is enriched for repeated sequences which are normally observed in restriction digests of P. chrysogenum total DNA. Conversely, type 2 appears to be composed entirely of non-repetitive sequences. Type 3 has been identified as mitochondrial DNA, having a buoyant density of 1.695 g/cm³ and an estimated molecular weight of 31.6×10⁶ Daltons.     

A comparison of the structures of apo dogfish M4 lactate dehydrogenase and its ternary complexes

Details are recorded of the X-ray diffraction data collection, heavy atom refinement and preliminary structure refinement for two different dogfish M₄ lactate dehydrogenase structures. One of these is the 2.0 Å resolution apoenzyme structure; the other is a 3.0 Å resolution abortive ternary complex. Two other ternary substrate inhibitory complexes (LDHase²: NAD: oxalate and LDHase: NADH: oxamate), isomorphous with the abortive ternary complex (LDHase: NAD-pyruvate), have also been examined. The apo-LDHase and LDHase: NAD-pyruvate structures are systematically compared to determine significant differences in their conformation. These are related to differences in structure amongst the three studied ternary complexes. These differences all occur in regions of the protein around the active site, particularly the flexible loop covering the active center pocket and the C-terminal helix αH. The changes are suggestive of a domino effect whereby the closing of the loop on binding coenzyme and substrate triggers the critical reactive residues into assuming their catalytically active positions.