Inhibitors of bacterial tyrosyl tRNA synthetase: synthesis of four stereoisomeric analogues of the natural product SB-219383

Synthetic analogues of the microbial metabolite SB-219383 have been synthesised with defined stereochemistry. Densely functionalised hydroxylamine containing amino acids were prepared by the addition of a glycine anion equivalent to sugar-derived cyclic nitrones. One of four stereoisomeric dipeptides incorporating these novel amino acids was found to be a potent and selective inhibitor of bacterial tyrosyl tRNA synthetase, suggesting analogous stereochemistry of the natural product.     

Evolution of a metabolic pathway for degradation of a toxic xenobiotic: the patchwork approach

The pathway for degradation of the xenobiotic pesticide pentachlorophenol in Sphingomonas chlorophenolica probably evolved in the past few decades by the recruitment of enzymes from two other catabolic pathways. The first and third enzymes in the pathway, pentachlorophenol hydroxylase and 2,6-dichlorohydroquinone dioxygenase, may have originated from enzymes in a pathway for degradation of a naturally occurring chlorinated phenol. The second enzyme, a reductive dehalogenase, may have evolved from a maleylacetoacetate isomerase normally involved in degradation of tyrosine. This apparently recently assembled pathway does not function very well: pentachlorophenol hydroxylase is quite slow, and tetrachlorohydroquinone dehalogenase is subject to severe substrate inhibition.     

Recent improvements to the SMART domain-based sequence annotation resource

SMART (Simple Modular Architecture Research Tool, http://smart.embl-heidelberg.de) is a web-based resource used for the annotation of protein domains and the analysis of domain architectures, with particular emphasis on mobile eukaryotic domains. Extensive annotation for each domain family is available, providing information relating to function, subcellular localization, phyletic distribution and tertiary structure. The January 2002 release has added more than 200 hand-curated domain models. This brings the total to over 600 domain families that are widely represented among nuclear, signalling and extracellular proteins. Annotation now includes links to the Online Mendelian Inheritance in Man (OMIM) database in cases where a human disease is associated with one or more mutations in a particular domain. We have implemented new analysis methods and updated others. New advanced queries provide direct access to the SMART relational database using SQL. This database now contains information on intrinsic sequence features such as transmembrane regions, coiled-coils, signal peptides and internal repeats. SMART output can now be easily included in users’ documents. A SMART mirror has been created at http://smart.ox.ac.uk.     

The reaction catalyzed by tetrachlorohydroquinone dehalogenase does not involve nucleophilic aromatic substitution.

Tetrachlorohydroquinone dehalogenase catalyzes the reductive dehalogenation of tetrachlorohydroquinone and trichlorohydroquinone during the biodegradation of the xenobiotic compound pentachlorophenol by Sphingobium chlorophenolicum. The mechanism of this transformation is of interest because it is unusual and difficult, and because aerobic microorganisms rarely catalyze reductive dehalogenation reactions. Tetrachlorohydroquinone dehalogenase is a member of the glutathione S-transferase superfamily. Many enzymes in this superfamily are capable of catalyzing nucleophilic aromatic substitution reactions. On the basis of this precedent, we have considered a mechanism for tetrachlorohydroquinone dehalogenase that involves a nucleophilic aromatic substitution reaction, either via an S(N)Ar mechanism or an S(RN)1-like mechanism, in the initial part of the reaction. Mechanistic studies were carried out with the wild type enzyme and with the C13S mutant enzyme, which catalyzes only the initial steps in the reaction. Three findings eliminate the possibility of a nucleophilic aromatic substitution reaction. First, the product of such a reaction, 2,3,5-trichloro-6-S-glutathionylhydroquinone, is not a kinetically competent intermediate. Second, the enzyme can carry out the reaction when the substrate is deprotonated at the active site. Nucleophilic aromatic substitution should not be possible when the substrate is negatively charged. Third, substantial normal solvent kinetic isotope effects on k(cat) and k(cat)/K(M,TriCHQ) are observed. Nonenzymatic and enzymatic nucleophilic S(N)Ar reactions typically show inverse solvent kinetic isotope effects.     

Amplicon Remodeling and Genomic Mutations Drive Population Dynamics after Segmental Amplification

New enzymes often evolve by duplication and divergence of genes encoding enzymes with promiscuous activities that have become important in the face of environmental opportunities or challenges. Amplifications that increase the copy number of the gene under selection commonly amplify many surrounding genes. Extra copies of these coamplified genes must be removed, either during or after evolution of a new enzyme. Here we report that amplicon remodeling can begin even before mutations occur in the gene under selection. Amplicon remodeling and mutations elsewhere in the genome that indirectly increase fitness result in complex population dynamics, leading to emergence of clones that have improved fitness by different mechanisms. In this work, one of the two most successful clones had undergone two episodes of amplicon remodeling, leaving only four coamplified genes surrounding the gene under selection. Amplicon remodeling in the other clone resulted in removal of 111 genes from the genome, an acceptable solution under these selection conditions, but one that would certainly impair fitness under other environmental conditions.     

Genome Shuffling Improves Degradation of the Anthropogenic Pesticide Pentachlorophenol by Sphingobium chlorophenolicum ATCC 39723

Pentachlorophenol (PCP), a highly toxic anthropogenic pesticide, can be mineralized by Sphingobium chlorophenolicum, a gram-negative bacterium isolated from PCP-contaminated soil. However, degradation of PCP is slow and S. chlorophenolicum cannot tolerate high levels of PCP. We have used genome shuffling to improve the degradation of PCP by S. chlorophenolicum. We have obtained several strains that degrade PCP faster and tolerate higher levels of PCP than the wild-type strain. Several strains obtained after the third round of shuffling can grow on one-quarter-strength tryptic soy broth plates containing 6 to 8 mM PCP, while the original strain cannot grow in the presence of PCP at concentrations higher than 0.6 mM. Some of the mutants are able to completely degrade 3 mM PCP in one-quarter-strength tryptic soy broth, whereas no degradation can be achieved by the wild-type strain. Analysis of several improved strains suggests that the improved phenotypes are due to various combinations of mutations leading to an enhanced growth rate, constitutive expression of the PCP degradation genes, and enhanced resistance to the toxicity of PCP and its metabolites.     

Effects of hematocrit on thixotropic properties of human blood

The rheological properties of whole human blood exhibit thixotropic behavior at low shear rates up to about ten reciprocal seconds (1). The accepted cause of this shear rate-dependent and time-dependent behavior is the progressive breakdown of rouleaux into individual red cells. Huang developed a rheological equation which incorporates the kinetics of rouleau breakdown in his models (2). This five-parameter equation was used successfully to represent the hysteresis loop and the torque-decay curve of whole human blood. Numerical values of these five thixotropic parameters, which characterize the rheological behavior of the blood from apparently healthy human subjects, were established (3). In this communication, we examined the effect of hematocrit on each of the above mentioned parameters. The results show that the following parameters will increase their values with an increase in hematocrit: the yield stress, Newtonian contribution of viscosity, non-Newtonian contribution of viscosity, apparent viscosity and the equilibrium value of the structural parameter which indicates the relative amount of rouleaux in blood. Mathematical equations were developed to give the relationship between parameters and hematocrit. Two other thixotropic parameters, viz. the kinetic rate constant of rouleaux breakdown into individual red cells and the order of the breakdown reaction, were found to be independent of the hematocrit. It is consistent with reaction kinetic theory that the rate constant and the order of reaction are independent of the concentration of reactants.     

Variation in structural location and amino acid conservation of functional sites in protein domain families

Background  

The functional sites of a protein present important information for determining its cellular function and are fundamental in drug design. Accordingly, accurate methods for the prediction of functional sites are of immense value. Most available methods are based on a set of homologous sequences and structural or evolutionary information, and assume that functional sites are more conserved than the average. In the analysis presented here, we have investigated the conservation of location and type of amino acids at functional sites, and compared the behaviour of functional sites between different protein domains.     

Animal phylogeny: fatal attraction

MPhylogenetic analyses of hundreds of genes from model animals have placed flies closer to vertebrates than to nematodes; recent work suggests this may be due to an artefact known as long branch attraction.     

MnPO4‐Coated Li(Ni0.4Co0.2Mn0.4)O2 for Lithium(‐Ion) Batteries with Outstanding Cycling Stability and Enhanced Lithiation Kinetics

Herein, the successful synthesis of MnPO₄‐coated LiNi_0.4Co_0.2Mn_0.4O₂ (MP‐NCM) as a lithium battery cathode material is reported. The MnPO₄ coating acts as an ideal protective layer, physically preventing the contact between the NCM active material and the electrolyte and, thus, stabilizing the electrode/electrolyte interface and preventing detrimental side reactions. Additionally, the coating enhances the lithium de‐/intercalation kinetics in terms of the apparent lithium‐ion diffusion coefficient. As a result, MP‐NCM‐based electrodes reveal greatly enhanced C‐rate capability and cycling stability—even under exertive conditions like extended operational potential windows, elevated temperature, and higher active material mass loadings. This superior electrochemical behavior of MP‐NCM compared to as‐synthesized NCM is attributed to the superior stability of the electrode/electrolyte interface and structural integrity when applying a MnPO₄ coating. Employing an ionic liquid as an alternative, intrinsically safer electrolyte system allows for outstanding cycling stabilities in a lithium‐metal battery configuration with a capacity retention of well above 85% after 2000 cycles. Similarly, the implementation in a lithium‐ion cell including a graphite anode provides stable cycling for more than 2000 cycles and an energy and power density of, respectively, 376 Wh kg^?1 and 1841 W kg^?1 on the active material level.