336 research outputs found
Structure-guided engineering of Lactococcus lactis alcohol dehydrogenase LlAdhA for improved conversion of isobutyraldehyde to isobutanol
We have determined the X-ray crystal structures of the NADH-dependent alcohol dehydrogenase LlAdhA from Lactococcus lactis and its laboratory-evolved variant LlAdhA^(RE1) at 1.9 Ă
and 2.5 Ă
resolution, respectively. LlAdhA^(RE1), which contains three amino acid mutations (Y50F, I212T, and L264V), was engineered to increase the microbial production of isobutanol (2-methylpropan-1-ol) from isobutyraldehyde (2-methylpropanal). Structural comparison of LlAdhA and LlAdhA^(RE1) indicates that the enhanced activity on isobutyraldehyde stems from increases in the protein's active site size, hydrophobicity, and substrate access. Further structure-guided mutagenesis generated a quadruple mutant (Y50F/N110S/I212T/L264V), whose K_M for isobutyraldehyde is âź17-fold lower and catalytic efficiency (k_(cat)/K_M) is âź160-fold higher than wild-type LlAdhA. Combining detailed structural information and directed evolution, we have achieved significant improvements in non-native alcohol dehydrogenase activity that will facilitate the production of next-generation fuels such as isobutanol from renewable resources
Mutating a Highly Conserved Residue in Diverse Cytochrome P450s Facilitates Diastereoselective Olefin Cyclopropanation
Cytochrome P450s and other heme-containing proteins have recently been shown to have promiscuous activity for the cyclopropanation of olefins using diazoacetate reagents. Despite the progress made thus far, engineering selective catalysts for all possible stereoisomers for the cyclopropanation reaction remains a considerable challenge. Previous investigations of a model P450 (P450BM3) revealed that mutation of a conserved active site threonine (Thr268) to alanine transformed the enzyme into a highly active and selective cyclopropanation catalyst. By incorporating this mutation into a diverse panel of P450 scaffolds, we were able to quickly identify enantioselective catalysts for all possible diastereomers in the model reaction of styrene with ethyl diazoacetate. Some alanine variants exhibited selectivities that were markedly different from the wild-type enzyme, with a few possessing moderate to high diastereoselectivity and enantioselectivities up to 97 % for synthetically challenging cis-cyclopropane diastereomers
Estado del Arte, y Perspectiva Actual del Concepto de Bienestar PsicolĂłGico en PsicologĂa del Deporte
Well-being psychology theory (Ryff, 1989) is one of the essential links of positive psychology, which has been mainly framed between two perspectives that explain a model of world and a model of person (DĂaz, 2006). Ryff (1989) states her model baptized as âIntegrated Model of Personal Developmentâ. Considering that sport, and life cycle sport are activities that demand to be strong, to have superior volitive skills, autonomy, be challenging, set up objectives, meet aims, grow as person, adapt to different environments and changes, and for that purpose, establish positive relationship with the others, be healthy and maintain self control, is for that reason we will stop into this concept and the analysis of psychological well-being and its involvement in sport, and sport psychology, from Carol Ryff perspective
Estado del arte, y perspectiva actual del concepto de bienestarpsicolĂłgico en psicologĂa del deporte
La TeorĂa del bienestar PsicolĂłgico (Ryff, 1989) constituye
uno de los eslabones fundamentales de la psicologĂa
positiva, la cual ha estado enmarcada principalmente entre
dos perspectivas que explican un modelo de mundo y un
modelo de sujeto (DĂaz, 2006). Ryff (1989) enuncia su
modelo bautizado como âModelo Integrado de
Desarrollo Personalâ (Integrated Model of Personal Develoment).
Considerando que el deporte y la vida deportiva son
actividades que requieren ser resistentes, alta capacidad
volitiva, autonomĂa, poseer desafĂos, plantearse objetivos,
cumplir metas, crecer como persona, adaptarse a los
distintos ambientes y cambios y para ello, relacionarse
positivamente con el entorno, ser saludable, y mantener
un control sobre lo que se hace, es por eso que nos
detendremos en el concepto, y el anĂĄlisis del bienestar
psicolĂłgico y sus implicancias en el deporte y la psicologĂa
del deporte, desde el enfoque de Carol Ryff
Vitamin D - a systematic literature review for the 5th edition of the Nordic Nutrition Recommendations
Peer reviewe
Soft drink and juice consumption and renal cell carcinoma incidence and mortality in the European Prospective Investigation into Cancer and Nutrition
Background: Renal cell carcinoma (RCC) accounts for more than 80% of kidney cancers in adults, and obesity is a known risk factor. Regular consumption of sweetened beverages has been linked to obesity and several chronic diseases, including some types of cancer. It is uncertain whether soft drink and juice consumption is associated with risk of RCC. We investigated the associations of soft drink and juice consumption with RCC incidence and mortality in the European Prospective Investigation into Cancer and Nutrition (EPIC). Methods: A total of 389,220 EPIC participants with median age of 52 years at recruitment (1991â2000) were included. Cox regression yielded adjusted HRs and 95% confidence intervals (CI) for RCC incidence and mortality in relation to intakes of juices and total, sugar-sweetened, and artificially sweetened soft drinks. Results: A total of 888 incident RCCs and 356 RCC deaths were identified. In models including adjustment for body mass index and energy intake, there was no higher risk of incident RCC associated with consumption of juices (HR per 100 g/day increment = 1.03; 95% CI, 0.97â1.09), total soft drinks (HR = 1.01; 95% CI, 0.98â1.05), sugar-sweetened soft drinks (HR = 0.99; 95% CI, 0.94â1.05), or artificially sweetened soft drinks (HR = 1.02; 95% CI, 0.96â1.08). In these fully adjusted models, none of the beverages was associated with RCC mortality (HR, 95% CI per 100 g/day increment 1.06, 0.97â1.16; 1.03, 0.98â1.09; 0.97, 0.89â1.07; and 1.06, 0.99â1.14, respectively). Conclusions: Consumption of juices or soft drinks was not associated with RCC incidence or mortality after adjusting for obesity
Genetic Incorporation of Unnatural Amino Acids into Proteins in Mycobacterium tuberculosis
New tools are needed to study the intracellular pathogen Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), to facilitate new drug discovery and vaccine development. We have developed methodology to genetically incorporate unnatural amino acids into proteins in Mycobacterium smegmatis, BCG and Mtb, grown both extracellularly in culture and inside host cells. Orthogonal mutant tRNATyr/tyrosyl-tRNA synthetase pairs derived from Methanococcus jannaschii and evolved in Escherichia coli incorporate a variety of unnatural amino acids (including photocrosslinking, chemically reactive, heavy atom containing, and immunogenic amino acids) into proteins in response to the amber nonsense codon. By taking advantage of the fidelity and suppression efficiency of the MjtRNA/pIpaRS pair in mycobacteria, we are also able to use p-iodophenylalanine to induce the expression of proteins in mycobacteria both extracellularly in culture and inside of mammalian host cells. This provides a new approach to regulate the expression of reporter genes or mycobacteria endogenous genes of interest. The establishment of the unnatural amino acid expression system in Mtb, an intracellular pathogen, should facilitate studies of TB biology and vaccine development
A serine-substituted P450 catalyzes highly efficient carbene transfer to olefins in vivo
Whole-cell catalysts for non-natural chemical reactions will open new routes to sustainable production of chemicals. We designed a cytochrome 'P411' with unique serine-heme ligation that catalyzes efficient and selective olefin cyclopropanation in intact Escherichia coli cells. The mutation C400S in cytochrome P450_(BM3) gives a signature ferrous CO Soret peak at 411 nm, abolishes monooxygenation activity, raises the resting-state FeIII-to-FeII reduction potential and substantially improves NAD(P)H-driven activity
Circulating concentrations of vitamin D in relation to pancreatic cancer risk in European populations
Evidence from in vivo, in vitro and ecological studies are suggestive of a protective effect of vitamin D against pancreatic cancer (PC). However, this has not been confirmed by analytical epidemiological studies. We aimed to examine the association between pre-diagnostic circulating vitamin D concentrations and PC incidence in European populations. We conducted a pooled nested case-control study within the European Prospective Investigation into Cancer and Nutrition (EPIC) and the Nord-Trøndelag Health Study's second survey (HUNT2) cohorts. In total, 738 primary incident PC cases (EPIC nâ=â626; HUNT2 nâ=â112; median follow-upâ=â6.9 years) were matched to 738 controls. Vitamin D [25(OH)D2 and 25(OH)D3 combined] concentrations were determined using isotope-dilution liquid chromatography-tandem mass spectrometry. Conditional logistic regression models with adjustments for body mass index and smoking habits were used to estimate incidence rate ratios (IRRs) and 95% confidence intervals (95%CI). Compared with a reference category of >50 to 75 nmol/L vitamin D, the IRRs (95% CIs) were 0.71 (0.42â1.20); 0.94 (0.72â1.22); 1.12 (0.82â1.53) and 1.26 (0.79â2.01) for clinically pre-defined categories of â¤25; >25 to 50; >75 to 100; and >100 nmol/L vitamin D, respectively (p for trendâ=â0.09). Corresponding analyses by quintiles of season-standardized vitamin D concentrations also did not reveal associations with PC risk (p for trendâ=â0.23). Although these findings among participants from the largest combination of European cohort studies to date show increasing effect estimates of PC risk with increasing pre-diagnostic concentrations of vitamin D, they are not statistically significant
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