Modifying the stereochemistry of an enzyme-catalyzed reaction by directed evolution

Aldolases have potential as tools for the synthesis of stereochemically complex carbohydrates. Here, we show that directed evolution can be used to alter the stereochemical course of the reaction catalyzed by tagatose-1,6-bisphosphate aldolase. After three rounds of DNA shuffling and screening, the evolved aldolase showed an 80-fold improvement in k-cat/K-m toward the non-natural substrate fructose 1,6-bisphosphate, resulting in a 100-fold change in stereospecificity. (31)P NMR spectroscopy was used to show that, in the synthetic direction, the evolved aldolase catalyzes the formation of carbon—carbon bonds with unnatural diastereoselectivity, where the >99:<1 preference for the formation of tagatose 1,6-bisphosphate was switched to a 4:1 preference for the diastereoisomer, fructose 1,6-bisphosphate. This demonstration is of considerable significance to synthetic chemists requiring efficient syntheses of complex stereoisomeric products, such as carbohydrate mimetics

False friends in the Fanfanyu

In the present article, a remarkable phenomenon is brought to the attention of those interested in early Chinese translations of Buddhist texts: false friends in the Fanfanyu (T54n2130). Baochang's Sanskrit-Chinese lexicon that was compiled as early as 517 AD reveals some curious examples of faux amis. In the present contribution, this case will be illustrated with references from the Shanjian lü piposha (T24n1462), a fifth century Chinese translation of the Samantapāsādikā, Buddhaghosa's commentary on the Pāli Vinaya. The fact that Baochang did not realise that this text was not translated from Sanskrit, inadvertently gave rise to some interesting jeux de mots

Somatostatin receptor expression, tumour response, and quality of life in patients with advanced hepatocellular carcinoma treated with long-acting octreotide

Octreotide may extend survival in hepatocellular carcinoma (HCC). Forty-one per cent of HCCs have high-affinity somatostatin receptors. We aimed to determine the feasibility, safety, and activity of long-acting octreotide in advanced HCC; to identify the best method for assessing somatostatin receptor expression; to relate receptor expression to clinical outcomes; and to evaluate toxicity. Sixty-three patients with advanced HCC received intramuscular long-acting octreotide 20 mg monthly until progression or toxicity. Median age was 67 years (range 28–81 years), male 81%, Child–Pugh A 83%, and B 17%. The aetiologies of chronic liver disease were alcohol (22%), viral hepatitis (44%), and haemochromatosis (6%). Prior treatments for HCC included surgery (8%), chemotherapy (2%), local ablation (11%), and chemoembolisation (6%). One patient had an objective partial tumour response (2%, 95% CI 0–9%). Serum alpha-fetoprotein levels decreased more than 50% in four (6%). Median survival was 8 months. Thirty four of 61 patients (56%) had receptor expression detected by scintigraphy; no clear relationship with clinical outcomes was identified. There were few grade 3 or 4 toxicities: hyperglycaemia (8%), hypoglycaemia (2%), diarrhoea (5%), and anorexia (2%). Patients reported improvements in some symptoms, but no major changes in quality of life were detected. Long-acting octreotide is safe in advanced HCC. We found little evidence of anticancer activity. A definitive randomised trial would identify whether patients benefit from this treatment in other ways

Introducing a new ICRU report: Prescribing, recording and reporting electron beam therapy

The ICRU published several Reports about volumes and doses specifications for radiotherapy, such as the Report 29 (1978), devoted to photon and electron beam therapy. This report 29 becoming absolete, a new Report was published in 1993 for external photon beam radiotherapy, the Report 50, recommending new definitions and more accurate specifications. With electran beams specific problems are raised, and the ICRU considered suitable to prepare a special Report for them, to be published in the near future.The main features of the present draft are as follows:1.Volumes specifications in agreement with the ICRU Report 50,•Volumes to be determined before treatment planning: gross tumour volume (GTV), c1inical target volume (CTV), organs at risk volumes (OR).•Volume to be determined during treatment planning: Planning target volume (PTV).•Volumes resulting fram the treatment plan chosen: treatment volume (TV), irradiated volume (IV).In the future Report on electron beams, an additional volume is defined, the internal target volume (ITV) geometrical concept representing the volume en-compassing the c1inical target volume, taking into consideration margins due to the variations of the clinical target volume in position, shape an size. A similar concept has been extended to organs at risk, the planning organ at risk volume.2.Dose specificationThe general statements for photon beams apply:•dose at a reference point (ICRU point) situated at or near the center of the planning target volume and, when possible, near or on the central axis of the electron beam at the depth of the peak dose.•Minimal and maximal doses in the planning target volume•Dose delivered to the organs at risk•Additional information is recommended, when possible (e.g. DVH).With electron beams, the dose homogeneity expected within the PTV (± 5 to ± 10 %) requires an adaptation of the terapeutic range concept, such that the value of the isodose surface encompassing the PTV be situated between 85 % and 95 % of the reference dose. The peak absorbed dose on the beam axis should always been specified, even if it is different fram the reference dose.At last, as in Report 50, three levels of dose evaluation for reporting are considered, depending on the aim of the treatment and the data available

Automated Alphabet Reduction for Protein Datasets

<p>Abstract</p> <p>Background</p> <p>We investigate automated and generic alphabet reduction techniques for protein structure prediction datasets. Reducing alphabet cardinality without losing key biochemical information opens the door to potentially faster machine learning, data mining and optimization applications in structural bioinformatics. Furthermore, reduced but informative alphabets often result in, e.g., more compact and human-friendly classification/clustering rules. In this paper we propose a robust and sophisticated alphabet reduction protocol based on mutual information and state-of-the-art optimization techniques.</p> <p>Results</p> <p>We applied this protocol to the prediction of two protein structural features: contact number and relative solvent accessibility. For both features we generated alphabets of two, three, four and five letters. The five-letter alphabets gave prediction accuracies statistically similar to that obtained using the full amino acid alphabet. Moreover, the automatically designed alphabets were compared against other reduced alphabets taken from the literature or human-designed, outperforming them. The differences between our alphabets and the alphabets taken from the literature were quantitatively analyzed. All the above process had been performed using a primary sequence representation of proteins. As a final experiment, we extrapolated the obtained five-letter alphabet to reduce a, much richer, protein representation based on evolutionary information for the prediction of the same two features. Again, the performance gap between the full representation and the reduced representation was small, showing that the results of our automated alphabet reduction protocol, even if they were obtained using a simple representation, are also able to capture the crucial information needed for state-of-the-art protein representations.</p> <p>Conclusion</p> <p>Our automated alphabet reduction protocol generates competent reduced alphabets tailored specifically for a variety of protein datasets. This process is done without any domain knowledge, using information theory metrics instead. The reduced alphabets contain some unexpected (but sound) groups of amino acids, thus suggesting new ways of interpreting the data.</p

γ-Aminobutyric Acid Transporter 2 Mediates the Hepatic Uptake of Guanidinoacetate, the Creatine Biosynthetic Precursor, in Rats

Guanidinoacetic acid (GAA) is the biosynthetic precursor of creatine which is involved in storage and transmission of phosphate-bound energy. Hepatocytes readily convert GAA to creatine, raising the possibility that the active uptake of GAA by hepatocytes is a regulatory factor. The purpose of this study is to investigate and identify the transporter responsible for GAA uptake by hepatocytes. The characteristics of [14C]GAA uptake by hepatocytes were elucidated using the in vivo liver uptake method, freshly isolated rat hepatocytes, an expression system of Xenopus laevis oocytes, gene knockdown, and an immunohistochemical technique. In vivo injection of [14C]GAA into the rat femoral vein and portal vein results in the rapid uptake of [14C]GAA by the liver. The uptake was markedly inhibited by γ-aminobutyric acid (GABA) and nipecotinic acid, an inhibitor of GABA transporters (GATs). The characteristics of Na+- and Cl−-dependent [14C]GAA uptake by freshly isolated rat hepatocytes were consistent with those of GAT2. The Km value of the GAA uptake (134 µM) was close to that of GAT2-mediated GAA transport (78.9 µM). GABA caused a marked inhibition with an IC50 value of 8.81 µM. The [14C]GAA uptake exhibited a significant reduction corresponding to the reduction in GAT2 protein expression. GAT2 was localized on the sinusoidal membrane of the hepatocytes predominantly in the periportal region. This distribution pattern was consistent with that of the creatine biosynthetic enzyme, S-adenosylmethionine∶guanidinoacetate N-methyltransferase. GAT2 makes a major contribution to the sinusoidal GAA uptake by periportal hepatocytes, thus regulating creatine biosynthesis in the liver

Role of Active Site Rigidity in Activity: MD Simulation and Fluorescence Study on a Lipase Mutant

Relationship between stability and activity of enzymes is maintained by underlying conformational flexibility. In thermophilic enzymes, a decrease in flexibility causes low enzyme activity while in less stable proteins such as mesophiles and psychrophiles, an increase in flexibility is associated with enhanced enzyme activity. Recently, we identified a mutant of a lipase whose stability and activity were enhanced simultaneously. In this work, we probed the conformational dynamics of the mutant and the wild type lipase, particularly flexibility of their active site using molecular dynamic simulations and time-resolved fluorescence techniques. In contrast to the earlier observations, our data show that active site of the mutant is more rigid than wild type enzyme. Further investigation suggests that this lipase needs minimal reorganization/flexibility of active site residues during its catalytic cycle. Molecular dynamic simulations suggest that catalytically competent active site geometry of the mutant is relatively more preserved than wild type lipase, which might have led to its higher enzyme activity. Our study implies that widely accepted positive correlation between conformation flexibility and enzyme activity need not be stringent and draws attention to the possibility that high enzyme activity can still be accomplished in a rigid active site and stable protein structures. This finding has a significant implication towards better understanding of involvement of dynamic motions in enzyme catalysis and enzyme engineering through mutations in active site

Molecular Dynamics Simulation of Phosphorylated KID Post-Translational Modification

BACKGROUND:Kinase-inducible domain (KID) as transcriptional activator can stimulate target gene expression in signal transduction by associating with KID interacting domain (KIX). NMR spectra suggest that apo-KID is an unstructured protein. After post-translational modification by phosphorylation, KID undergoes a transition from disordered to well folded protein upon binding to KIX. However, the mechanism of folding coupled to binding is poorly understood. METHODOLOGY:To get an insight into the mechanism, we have performed ten trajectories of explicit-solvent molecular dynamics (MD) for both bound and apo phosphorylated KID (pKID). Ten MD simulations are sufficient to capture the average properties in the protein folding and unfolding. CONCLUSIONS:Room-temperature MD simulations suggest that pKID becomes more rigid and stable upon the KIX-binding. Kinetic analysis of high-temperature MD simulations shows that bound pKID and apo-pKID unfold via a three-state and a two-state process, respectively. Both kinetics and free energy landscape analyses indicate that bound pKID folds in the order of KIX access, initiation of pKID tertiary folding, folding of helix alpha(B), folding of helix alpha(A), completion of pKID tertiary folding, and finalization of pKID-KIX binding. Our data show that the folding pathways of apo-pKID are different from the bound state: the foldings of helices alpha(A) and alpha(B) are swapped. Here we also show that Asn139, Asp140 and Leu141 with large Phi-values are key residues in the folding of bound pKID. Our results are in good agreement with NMR experimental observations and provide significant insight into the general mechanisms of binding induced protein folding and other conformational adjustment in post-translational modification

Mitochondrial Genome Sequences Effectively Reveal the Phylogeny of Hylobates Gibbons

BACKGROUND: Uniquely among hominoids, gibbons exist as multiple geographically contiguous taxa exhibiting distinctive behavioral, morphological, and karyotypic characteristics. However, our understanding of the evolutionary relationships of the various gibbons, especially among Hylobates species, is still limited because previous studies used limited taxon sampling or short mitochondrial DNA (mtDNA) sequences. Here we use mtDNA genome sequences to reconstruct gibbon phylogenetic relationships and reveal the pattern and timing of divergence events in gibbon evolutionary history. METHODOLOGY/PRINCIPAL FINDINGS: We sequenced the mitochondrial genomes of 51 individuals representing 11 species belonging to three genera (Hylobates, Nomascus and Symphalangus) using the high-throughput 454 sequencing system with the parallel tagged sequencing approach. Three phylogenetic analyses (maximum likelihood, Bayesian analysis and neighbor-joining) depicted the gibbon phylogenetic relationships congruently and with strong support values. Most notably, we recover a well-supported phylogeny of the Hylobates gibbons. The estimation of divergence times using Bayesian analysis with relaxed clock model suggests a much more rapid speciation process in Hylobates than in Nomascus. CONCLUSIONS/SIGNIFICANCE: Use of more than 15 kb sequences of the mitochondrial genome provided more informative and robust data than previous studies of short mitochondrial segments (e.g., control region or cytochrome b) as shown by the reliable reconstruction of divergence patterns among Hylobates gibbons. Moreover, molecular dating of the mitogenomic divergence times implied that biogeographic change during the last five million years may be a factor promoting the speciation of Sundaland animals, including Hylobates species