Nitrile hydratases (NHases) are important biocatalysts for the enzymatic conversion of nitriles to industrially-important amides such as acrylamide and nicotinamide. Although thermostability in this ...enzyme class is generally low, there is not sufficient understanding of its basis for rational enzyme design. The gene expressing the Co-type NHase from the moderate thermophile, Geobacillus pallidus RAPc8 (NRRL B-59396), was subjected to random mutagenesis. Four mutants were selected that were 3 to 15-fold more thermostable than the wild-type NHase, resulting in a 3.4–7.6 kJ/mol increase in the activation energy of thermal inactivation at 63 °C. High resolution X-ray crystal structures (1.15–1.80 Å) were obtained of the wild-type and four mutant enzymes. Mutant 9E, with a resolution of 1.15 Å, is the highest resolution crystal structure obtained for a nitrile hydratase to date. Structural comparisons between the wild-type and mutant enzymes illustrated the importance of salt bridges and hydrogen bonds in enhancing NHase thermostability. These additional interactions variously improved thermostability by increased intra- and inter-subunit interactions, preventing cooperative unfolding of α-helices and stabilising loop regions. Some hydrogen bonds were mediated via a water molecule, specifically highlighting the significance of structured water molecules in protein thermostability. Although knowledge of the mutant structures makes it possible to rationalize their behaviour, it would have been challenging to predict in advance that these mutants would be stabilising.
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•Random mutagenesis yields a 15-fold increase in nitrile hydratase thermostability.•Salt bridges and hydrogen bonds improves nitrile hydratase thermostability.•Water-mediated hydrogen bonds improves protein thermostability.
Summary Aim To determine the clinical utility of a rapid molecular assay for Clostridium difficile infection (CDI) in an acute hospital setting. Methods From March to September 2011, stool specimens ...from inpatients in two acute hospitals with suspected CDI were tested prospectively by routine cell culture cytotoxin neutralization assay (CCNA), real-time polymerase chain reaction (PCR) using the GeneXpert (Cepheid Inc., Sunnyvale, CA, USA), and a dual testing algorithm glutamate dehydrogenase (GDH)/toxin enzyme immuno-assay, Premier, Launch Diagnostics, Longfield, UK. All patients with positive PCR, CCNA or discrepant results were reviewed by a multi-disciplinary team (treating clinician, gastroenterologist, microbiologist and infection control nurse). Results C. difficile detection rates were 11.7% (PCR), 6% (CCNA) and 13.8% (GDH). Out of 1034 stool specimens included in the study, 974 (94.1%) had concordant CCNA and PCR results. Eighty-nine percent (886/985) had concordant CCNA, PCR and GDH results, and 94.4% (930/985) had concordant GDH and PCR results. Using clinical diagnosis as the reference, PCR had sensitivity of 99.1%, specificity of 98.9%, positive predictive value (PPV) of 91.9% and negative predictive value (NPV) of 99.9%. CCNA on a single sample had sensitivity of 51%, specificity of 99.4%, PPV of 91.9% and NPV of 94.3%. GDH had sensitivity of 83.8%, specificity of 94.5%, PPV of 64.7% and NPV of 97.9%. Almost twice as many patients were positive by PCR compared with CCNA (121 vs 62); 54/59 of those with discrepant results were clinically confirmed as CDI. Conclusion Rapid diagnosis of CDI using PCR was timely, accurate and correlated well with clinical diagnosis.
Promoting the kinetics of CO2 hydrate formation using additives is of great importance in industrial applications of gas hydrates such as capture and storage of carbon dioxide. However, the mechanism ...of hydrate formation in the presence of solid particles is not well understood. This paper aims to gain a better understanding of the fundamental aspects of CO2 hydrates formation in the presence of hydrophobic silica nanoparticles. A novel mechanism for gas hydrate formation in the presence of hydrophobic particles was established from a series of well-designed experiments. Two types of particle stabilized systems, made by mixing hydrophobic silica nanoparticles and water, were tested: air-in-water foam and water-in-air powder (dry water). Gas consumption, CO2 conversion, induction time, and hydrate growth rate have been examined to establish the influence of particle hydrophobicity and concentration on hydrate formation kinetics. The results show that the promoting effect depends on the particle hydrophobicity and concentration. The most hydrophobic silica (dry water) enhances the kinetics of CO2 hydrate formation effectively. Cryogenic scanning electron microscopy, combined with energy-dispersive spectroscopy, was used to examine the morphology, microstructure, and pore characteristics of CO2 gas hydrates, as well as elemental composition of the samples. To provide further insight into the adsorption of gas molecules at the water/solid interface, surface potential of the hydrophobic fumed silica particles in aqueous system before and after exposing to N2 and CO2 gas was measured. These results, in combination with the result of our recent study on the structure of dry water, successfully provided further detailed information on how hydrophobic fumed silica promotes the formation of gas hydrates.
Cyanide dihydratase is an enzyme in the nitrilase family capable of transforming cyanide to formate and ammonia. This reaction has been exploited for the bioremediation of cyanide in wastewater ...streams, but extending the pH operating range of the enzyme would improve its utility. In this work, we describe mutants of
Bacillus pumilus
C1 cyanide dihydratase (CynD
pum
) with improved activity at higher pH. Error-prone PCR was used to construct a library of CynD
pum
mutants, and a high-throughput screening system was developed to screen the library for improved activity at pH 10. Two mutant alleles were identified that allowed cells to degrade cyanide in solutions at pH 10, whereas the wild-type was inactive above pH 9. The mutant alleles each encoded three different amino acid substitutions, but for one of those, a single change, E327G, accounted for the phenotype. The purified proteins containing multiple mutations were five times more active than the wild-type enzyme at pH 9, but all purified enzymes lost activity at pH 10. The mutation Q86R resulted in the formation of significantly longer fibers at low pH, and both E327G and Q86R contributed to the persistence of active oligomeric assemblies at pH 9. In addition, the mutant enzymes proved to be more thermostable than the wild type, suggesting improved physical stability rather than any change in chemistry accounts for their increased pH tolerance.
Angiotensin I-converting enzyme (ACE) is central to the regulation of the renin−angiotensin system and is a key therapeutic target for combating hypertension and related cardiovascular diseases. ...Currently available drugs bind both active sites of its two homologous domains, although it is now understood that these domains function differently in vivo. The recently solved crystal structures of both domains (N and C) open the door to new domain-specific inhibitor design, taking advantage of the differences between these two large active sites. Here we present the first crystal structure at a resolution of 2.25 Å of testis ACE (identical to the C domain of somatic ACE) with the highly C-domain-specific phosphinic inhibitor, RXPA380. Testis ACE retains the same conformation as seen in previously determined inhibitor complexes, but the RXPA380 central backbone conformation is more similar to that seen for the inhibitor captopril than enalaprilat. The RXPA380 molecule occupies more subsites of the testis ACE active site than the previously determined inhibitors and possesses bulky moieties that extend into the S2‘ and S2 subsites. Thus the high affinity of RXPA380 for the testis ACE/somatic ACE C domain is explained by the interaction of these bulky moieties with residues unique to these domains, specifically Phe 391, Val 379, and Val 380, that are not found in the N domain. The characterization of the extended active site and the binding of a potent C-domain-selective inhibitor provide the first structural data for the design of truly domain-specific pharmacophores.
Emulsions of monopalmitoylglycerol (MPG) and of a neutral lipid blend (NLB), consisting of MPG, monostearoylglycerol, dipalmitoylglycerol, dioleoylglycerol, and dilineoylglycerol (4:2:1:1:1), the ...composition associated with hemozoin from the malaria parasite Plasmodium falciparum, have been used to mediate the formation of β-hematin microcrystals. Transmission electron microscopy (TEM), electron diffraction, and electron spectroscopic imaging/electron energy loss spectroscopy (ESI/EELS) have been used to characterize both the lipid emulsion and β-hematin crystals. The latter have been compared with β-hematin formed at a pentanol/aqueous interface and with hemozoin both within P. falciparum parasites and extracted from the parasites. When lipid and ferriprotoporphyrin IX solutions in 1:9 v/v acetone/methanol were thoroughly premixed either using an extruder or an ultrasound bath, β-hematin crystals were found formed in intimate association with the lipid droplets. These crystals resembled hemozoin crystals, with prominent {100} faces. Lattice fringes in TEM indicated that these faces made contact with the lipid surface. The average length of these crystals was 0.62 times the average diameter of NLB droplets, and their size distributions were statistically equivalent after 10 min incubation, suggesting that the lipid droplets also controlled the sizes of the crystals. This most closely resembles hemozoin formation in the helminth worm Schistosoma mansoni, while in P. falciparum, crystal formation appears to be associated with the much more gently curved digestive vacuole membrane, which apparently leads to formation of much larger hemozoin crystals, similar to those formed at the flat pentanol–water interface.
Human ACE (angiotensin-converting enzyme) (EC 3.4.15.1) is an important drug target because of its role in the regulation of blood pressure via the renin-angiotensin-aldosterone system. Somatic ACE ...comprises two homologous domains, the differing substrate preferences of which present a new avenue for domain-selective inhibitor design. We have co-crystallized lisW-S, a C-domain-selective derivative of the drug lisinopril, with human testis ACE and determined a structure using X-ray crystallography to a resolution of 2.30 A (1 A=0.1 nm). In this structure, lisW-S is seen to have a similar binding mode to its parent compound lisinopril, but the P2' tryptophan moiety takes a different conformation to that seen in other inhibitors having a tryptophan residue in this position. We have examined further the domain-specific interactions of this inhibitor by mutating C-domain-specific active-site residues to their N domain equivalents, then assessing the effect of the mutation on inhibition by lisW-S using a fluorescence-based assay. Kinetics analysis shows a 258-fold domain-selectivity that is largely due to the co-operative effect of C-domain-specific residues in the S2' subsite. The high affinity and selectivity of this inhibitor make it a good lead candidate for cardiovascular drug development.
Nitrilases pose attractive alternatives to the chemical hydrolysis of nitrile compounds. The activity of bacterial nitrilases towards substrate is intimately tied to the formation of large ...spiral-shaped oligomers. In the nitrilase CynD (cyanide dihydratase) from
, mutations in a predicted oligomeric surface region altered its oligomerization and reduced its activity. One mutant, CynD Y70C, retained uniform oligomer formation however it was inactive, unlike all other inactive mutants throughout that region all of which significantly perturbed oligomer formation. It was hypothesized that Y70 is playing an additional role necessary for CynD activity beyond influencing oligomerization. Here, we performed saturation mutagenesis at residue 70 and demonstrated that only tyrosine or phenylalanine is permissible for CynD activity. Furthermore, we show that other residues at this position are not only inactive, but have altered or disrupted oligomer conformations. These results suggest that Y70's essential role in activity is independent of its role in the formation of the spiral oligomer.
ObjectiveWe sought to conduct a systematic review and meta-analysis of evidence to inform policies that reduce density and proximity of tobacco retailers.Data sourcesTen databases were searched on 16 ...October 2020: MEDLINE via PubMed, PsycINFO, Global Health, LILACS, Embase, ABI/Inform, CINAHL, Business Source Complete, Web of Science and Scopus, plus grey literature searches using Google and the RAND Publication Database.Study selectionIncluded studies used inferential statistics about adult participants to examine associations between tobacco retailer density/proximity and tobacco use behaviours and health outcomes. Of 7373 studies reviewed by independent coders, 37 (0.5%) met inclusion criteria.Data extractionEffect sizes were converted to a relative risk reduction (RRR) metric, indicating the presumed reduction in tobacco use outcomes based on reducing tobacco retailer density and decreasing proximity.Data synthesisWe conducted a random effects meta-analysis and examined heterogeneity across 27 studies through subgroup analyses and meta-regression. Tobacco retailer density (RRR=2.55, 95% CI 1.91 to 3.19, k=155) and proximity (RRR=2.38, 95% CI 1.39 to 3.37, k=100) were associated with tobacco use behaviours. Pooled results including both density and proximity found an estimated 2.48% reduction in risk of tobacco use from reductions in tobacco retailer density and proximity (RRR=2.48, 95% CI 1.95 to 3.02, k=255). Results for health outcomes came from just two studies and were not significant. Considerable heterogeneity existed.ConclusionsAcross studies, lower levels of tobacco retailer density and decreased proximity are associated with lower tobacco use. Reducing tobacco supply by limiting retailer density and proximity may lead to reductions in tobacco use. Policy evaluations are needed.
Critically ill patients often develop large gastric residual volumes during nasogastric feeding as a result of poor gastroduodenal motility. Nasojejunal feeding may decrease the severity of this ...complication. The aim of this study was to determine whether nasojejunal feeding improved tolerance of enteral nutrition by reducing gastric residual volumes.
Randomized, prospective, clinical study.
Intensive care unit of a university-affiliated hospital.
Seventy-three intensive care unit patients expected to require nutritional support for at least 3 days.
Patients were randomized to receive enteral nutrition via a nasojejunal tube (placed endoscopically) (34 patients) or a nasogastric tube (39 patients). A strict protocol was followed, which included regular gastric residual volume measurement (in both groups), the use of predetermined criteria for intolerance, and an attempt at nasojejunal feeding for those nasogastrically fed patients who were intolerant of enteral nutrition.
Endoscopic placement of nasojejunal tubes was successful in 98% with no complications of insertion. Patients fed via a nasojejunal tube had 1) a reduced total gastric residual volume in both the first 24 (197 vs. 491 mL, p = .02) and 48 hrs (517 vs. 975 mL, p = .02); 2) a reduced incidence of a single gastric residual volume >150 mL (32% vs. 74%, p = .001); and 3) a trend toward a reduced incidence of intolerance of enteral nutrition (13% vs. 31%, p = .09). Only 13% of those nasogastrically fed patients who were initially intolerant of enteral nutrition remained intolerant once fed via a nasojejunal tube, and only 1.4% of all patients met criteria for commencement of parenteral nutrition.
Enteral nutrition delivered via a nasojejunal tube is associated with a significant reduction in gastric residual volume, a strong trend toward improved tolerance of enteral nutrition, and an extremely low requirement for parenteral nutrition.