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‣ Evaluating the effects of a single amino acid substitution on both the native and denatured states of a protein.

Lin, T Y; Kim, P S
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/12/1991 Português
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For proteins that contain a disulfide bond, stability is linked thermodynamically to thiol-disulfide exchange. We use this relationship to obtain unfolding free energies for both the reduced and oxidized forms of Escherichia coli thioredoxin from measurements of the effective concentrations of protein thiols. We then evaluate the effect of an amino acid substitution on disulfide bond formation in both the native and denatured states of the protein. Although the Pro-34----Ser substitution in thioredoxin results in a decrease of the effective concentration of protein thiols in the native state, the effective concentration increases in the denatured state. The net effect of the amino acid substitution is to increase the stability of reduced thioredoxin by approximately 2.4 kcal/mol, whereas the stability of the oxidized protein remains the same. By assuming a two-state unfolding equilibrium and a mutation free energy of -7.7 kcal/mol for the Pro-34----Ser substitution in the reduced, urea-unfolded state (based on estimates of solvation and entropic changes), we obtained relative free energies for the native and denatured states of the mutant and wild-type proteins, in both the reduced and oxidized forms.

‣ A Single Amino Acid Substitution in a Mannosyltransferase, WbdA, Converts the Escherichia coli O9 Polysaccharide into O9a: Generation of a New O-Serotype Group

Kido, Nobuo; Kobayashi, Hidemitsu
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /05/2000 Português
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wbdA is a mannosyltransferase gene that is involved in synthesis of the Escherichia coli O9a polysaccharide, a mannose homopolymer with a repeating unit of 2-αMan-1,2-αMan-1,3-αMan-1,3-αMan-1. The equivalent structural O polysaccharide in the E. coli O9 and Klebsiella O3 strains is 2-αMan-1,2-αMan-1,2-αMan-1,3-αMan-1,3-αMan-1, with an excess of one mannose in the 1,2 linkage. We have cloned wbdA genes from these O9 and O3 strains and shown by genetic and functional studies that wbdA is the only gene determining the O-polysaccharide structure of O9 or O9a. Based on functional analysis of chimeric genes and site-directed mutagenesis, we showed that a single amino acid substitution, C55R, in WbdA of E. coli O9 converts the O9 polysaccharide into O9a. DNA sequencing revealed the substitution to be conserved in other E. coli O9a strains. The reverse substitution, R55C, in WbdA of E. coli O9a resulted in lipopolysaccharide synthesis showing no ladder profile instead of the conversion of O9a to O9. This suggests that more than one amino acid substitution in WbdA is required for conversion from O9a to O9.

‣ A naturally occurring single basic amino acid substitution in the V3 region of the human immunodeficiency virus type 1 env protein alters the cellular host range and antigenic structure of the virus.

Shioda, T; Oka, S; Ida, S; Nokihara, K; Toriyoshi, H; Mori, S; Takebe, Y; Kimura, S; Shimada, K; Nagai, Y
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /12/1994 Português
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Human immunodeficiency virus type 1 circulates in vivo as a mixture of heterologous populations (quasispecies). We previously analyzed the quasispecies of the third hypervariable region (V3) in the viral envelope glycoprotein gp120 in an infected individual and found that the species with a basic amino acid substitution (lysine for aspartic acid) at a particular position evolved and became a distinct population within a short period, followed by progression to the typical immunodeficiency stage (S. Oka et al., AIDS Res. Hum. Retroviruses 10:271-277, 1994). In the present study, we examined the biological significance of this amino acid substitution by constructing recombinant viruses with specific point mutations and comparing their replication capabilities in different cell types. The results demonstrated that the single basic amino acid substitution was sufficient to render a virus fully capable of replicating in human T-cell lines under certain conditions. With an acidic amino acid at the position, the virus grew much less fast or did not grow at all in the T-cell lines. Viral neutralization assay and peptide enzyme-linked immunosorbent assays further showed that this amino acid substitution resulted in different recognition by several of the serum specimens from human immunodeficiency virus type 1-infected individuals and thus could alter the antigenic structure. An additional finding worthy of note was that at the terminal stage...

‣ The compositional adjustment of amino acid substitution matrices

Yu, Yi-Kuo; Wootton, John C.; Altschul, Stephen F.
Fonte: National Academy of Sciences Publicador: National Academy of Sciences
Tipo: Artigo de Revista Científica
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Amino acid substitution matrices are central to protein-comparison methods. In most commonly used matrices, the substitution scores take a log-odds form, involving the ratio of “target” to “background” frequencies derived from large, carefully curated sets of protein alignments. However, such matrices often are used to compare protein sequences with amino acid compositions that differ markedly from the background frequencies used for the construction of the matrices. Of course, the target frequencies should be adjusted in such cases, but the lack of an appropriate way to do this has been a long-standing problem. This article shows that if one demands consistency between target and background frequencies, then a log-odds substitution matrix implies a unique set of target and background frequencies as well as a unique scale. Standard substitution matrices therefore are truly appropriate only for the comparison of proteins with standard amino acid composition. Accordingly, we present and evaluate a rationale for transforming the target frequencies implicit in a standard matrix to frequencies appropriate for a nonstandard context. This rationale yields asymmetric matrices for the comparison of proteins with divergent compositions. Earlier approaches are unable to deal with this case in a fully consistent manner. Composition-specific substitution matrix adjustment is shown to be of utility for comparing compositionally biased proteins...

‣ Alignment of 700 globin sequences: extent of amino acid substitution and its correlation with variation in volume.

Kapp, O. H.; Moens, L.; Vanfleteren, J.; Trotman, C. N.; Suzuki, T.; Vinogradov, S. N.
Fonte: Cold Spring Harbor Laboratory Press Publicador: Cold Spring Harbor Laboratory Press
Tipo: Artigo de Revista Científica
Publicado em /10/1995 Português
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Seven-hundred globin sequences, including 146 nonvertebrate sequences, were aligned on the basis of conservation of secondary structure and the avoidance of gap penalties. Of the 182 positions needed to accommodate all the globin sequences, only 84 are common to all, including the absolutely conserved PheCD1 and HisF8. The mean number of amino acid substitutions per position ranges from 8 to 13 for all globins and 5 to 9 for internal positions. Although the total sequence volumes have a variation approximately 2-3%, the variation in volume per position ranges from approximately 13% for the internal to approximately 21% for the surface positions. Plausible correlations exist between amino acid substitution and the variation in volume per position for the 84 common and the internal but not the surface positions. The amino acid substitution matrix derived from the 84 common positions was used to evaluate sequence similarity within the globins and between the globins and phycocyanins C and colicins A, via calculation of pairwise similarity scores. The scores for globin-globin comparisons over the 84 common positions overlap the globin-phycocyanin and globin-colicin scores, with the former being intermediate. For the subset of internal positions...

‣ Bayesian analysis of amino acid substitution models

Huelsenbeck, John P; Joyce, Paul; Lakner, Clemens; Ronquist, Fredrik
Fonte: The Royal Society Publicador: The Royal Society
Tipo: Artigo de Revista Científica
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Models of amino acid substitution present challenges beyond those often faced with the analysis of DNA sequences. The alignments of amino acid sequences are often small, whereas the number of parameters to be estimated is potentially large when compared with the number of free parameters for nucleotide substitution models. Most approaches to the analysis of amino acid alignments have focused on the use of fixed amino acid models in which all of the potentially free parameters are fixed to values estimated from a large number of sequences. Often, these fixed amino acid models are specific to a gene or taxonomic group (e.g. the Mtmam model, which has parameters that are specific to mammalian mitochondrial gene sequences). Although the fixed amino acid models succeed in reducing the number of free parameters to be estimated—indeed, they reduce the number of free parameters from approximately 200 to 0—it is possible that none of the currently available fixed amino acid models is appropriate for a specific alignment. Here, we present four approaches to the analysis of amino acid sequences. First, we explore the use of a general time reversible model of amino acid substitution using a Dirichlet prior probability distribution on the 190 exchangeability parameters. Second...

‣ Lineage-Specific Differences in the Amino Acid Substitution Process

Huzurbazar, Snehalata; Kolesov, Grigory; Massey, Steven E.; Harris, Katherine C.; Churbanov, Alexander; Liberles, David A.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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In Darwinian evolution, mutations occur approximately at random in a gene, turned into amino acid mutations by the genetic code. Some mutations are fixed to become substitutions and some are eliminated from the population. Partitioning pairs of closely related species with complete genome sequences by average population size of each pair, we looked at the substitution matrices generated for these partitions and compared the substitution patterns between species. We estimated a population genetic model that relates the relative fixation probabilities of different types of mutations to the selective pressure and population size. Parameterizations of the average and distribution of selective pressures for different amino acid substitution types in different population size comparisons were generated with a Bayesian framework. We found that partitions in population size as well as in substitution type are required to explain the substitution data. Selection coefficients were found to decrease with increasingly radical amino acid substitution and with increasing effective population size.

‣ CodonTest: Modeling Amino Acid Substitution Preferences in Coding Sequences

Delport, Wayne; Scheffler, Konrad; Botha, Gordon; Gravenor, Mike B.; Muse, Spencer V.; Kosakovsky Pond, Sergei L.
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
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Codon models of evolution have facilitated the interpretation of selective forces operating on genomes. These models, however, assume a single rate of non-synonymous substitution irrespective of the nature of amino acids being exchanged. Recent developments have shown that models which allow for amino acid pairs to have independent rates of substitution offer improved fit over single rate models. However, these approaches have been limited by the necessity for large alignments in their estimation. An alternative approach is to assume that substitution rates between amino acid pairs can be subdivided into rate classes, dependent on the information content of the alignment. However, given the combinatorially large number of such models, an efficient model search strategy is needed. Here we develop a Genetic Algorithm (GA) method for the estimation of such models. A GA is used to assign amino acid substitution pairs to a series of rate classes, where is estimated from the alignment. Other parameters of the phylogenetic Markov model, including substitution rates, character frequencies and branch lengths are estimated using standard maximum likelihood optimization procedures. We apply the GA to empirical alignments and show improved model fit over existing models of codon evolution. Our results suggest that current models are poor approximations of protein evolution and thus gene and organism specific multi-rate models that incorporate amino acid substitution biases are preferred. We further anticipate that the clustering of amino acid substitution rates into classes will be biologically informative...

‣ Impact of the E540V Amino Acid Substitution in GyrB of Mycobacterium tuberculosis on Quinolone Resistance▿†

Kim, Hyun; Nakajima, Chie; Yokoyama, Kazumasa; Rahim, Zeaur; Kim, Youn Uck; Oguri, Hiroki; Suzuki, Yasuhiko
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /08/2011 Português
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Amino acid substitutions conferring resistance to quinolones in Mycobacterium tuberculosis have generally been found within the quinolone resistance-determining regions (QRDRs) in the A subunit of DNA gyrase (GyrA) rather than the B subunit of DNA gyrase (GyrB). To clarify the contribution of an amino acid substitution, E540V, in GyrB to quinolone resistance in M. tuberculosis, we expressed recombinant DNA gyrases in Escherichia coli and characterized them in vitro. Wild-type and GyrB-E540V DNA gyrases were reconstituted in vitro by mixing recombinant GyrA and GyrB. Correlation between the amino acid substitution and quinolone resistance was assessed by the ATP-dependent DNA supercoiling assay, quinolone-inhibited supercoiling assay, and DNA cleavage assay. The 50% inhibitory concentrations of eight quinolones against DNA gyrases bearing the E540V amino acid substitution in GyrB were 2.5- to 36-fold higher than those against the wild-type enzyme. Similarly, the 25% maximum DNA cleavage concentrations were 1.5- to 14-fold higher for the E540V gyrase than for the wild-type enzyme. We further demonstrated that the E540V amino acid substitution influenced the interaction between DNA gyrase and the substituent(s) at R-7, R-8, or both in quinolone structures. This is the first detailed study of the contribution of the E540V amino acid substitution in GyrB to quinolone resistance in M. tuberculosis.

‣ Evaluating the efficacy of a structure-derived amino acid substitution matrix in detecting protein homologs by BLAST and PSI-BLAST

Goonesekere, Nalin CW
Fonte: Dove Medical Press Publicador: Dove Medical Press
Tipo: Artigo de Revista Científica
Publicado em 05/06/2009 Português
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The large numbers of protein sequences generated by whole genome sequencing projects require rapid and accurate methods of annotation. The detection of homology through computational sequence analysis is a powerful tool in determining the complex evolutionary and functional relationships that exist between proteins. Homology search algorithms employ amino acid substitution matrices to detect similarity between proteins sequences. The substitution matrices in common use today are constructed using sequences aligned without reference to protein structure. Here we present amino acid substitution matrices constructed from the alignment of a large number of protein domain structures from the structural classification of proteins (SCOP) database. We show that when incorporated into the homology search algorithms BLAST and PSI-blast, the structure-based substitution matrices enhance the efficacy of detecting remote homologs.

‣ Emergence of Avian Influenza Viruses with Enhanced Transcription Activity by a Single Amino Acid Substitution in the Nucleoprotein during Replication in Chicken Brains ▿

Tada, Tatsuya; Suzuki, Koutaro; Sakurai, Yu; Kubo, Masanori; Okada, Hironao; Itoh, Toshihiro; Tsukamoto, Kenji
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /10/2011 Português
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To explore the genetic basis of the pathogenesis and adaptation of avian influenza viruses (AIVs) to chickens, the A/duck/Yokohama/aq10/2003 (H5N1) (DkYK10) virus was passaged five times in the brains of chickens. The brain-passaged DkYK10-B5 caused quick death of chickens through rapid and efficient replication in tissues, accompanied by severe apoptosis. Genome sequence comparison of two viruses identified a single amino acid substitution at position 109 in NP from isoleucine to threonine (NP I109T). By analyzing viruses constructed by the reverse-genetic method, we established that the NP I109T substitution also contributed to increased viral replication and polymerase activity in chicken embryo fibroblasts, but not in duck embryo fibroblasts. Real-time RT-PCR analysis demonstrated that the NP I109T substitution enhances mRNA synthesis quickly and then cRNA and viral RNA (vRNA) synthesis slowly. Next, to determine the mechanism underlying the appearance of the NP I109T substitution during passages, four H5N1 highly pathogenic AIVs (HPAIVs) were passaged in the lungs and brains of chicken embryos. Single-nucleotide polymorphism analysis, together with a database search, suggests that the NP I109T mutation would be induced frequently during replication of HPAIVs in brains...

‣ Antibody-Specific Model of Amino Acid Substitution for Immunological Inferences from Alignments of Antibody Sequences

Mirsky, Alexander; Kazandjian, Linda; Anisimova, Maria
Fonte: Oxford University Press Publicador: Oxford University Press
Tipo: Artigo de Revista Científica
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Antibodies are glycoproteins produced by the immune system as a dynamically adaptive line of defense against invading pathogens. Very elegant and specific mutational mechanisms allow B lymphocytes to produce a large and diversified repertoire of antibodies, which is modified and enhanced throughout all adulthood. One of these mechanisms is somatic hypermutation, which stochastically mutates nucleotides in the antibody genes, forming new sequences with different properties and, eventually, higher affinity and selectivity to the pathogenic target. As somatic hypermutation involves fast mutation of antibody sequences, this process can be described using a Markov substitution model of molecular evolution. Here, using large sets of antibody sequences from mice and humans, we infer an empirical amino acid substitution model AB, which is specific to antibody sequences. Compared with existing general amino acid models, we show that the AB model provides significantly better description for the somatic evolution of mice and human antibody sequences, as demonstrated on large next generation sequencing (NGS) antibody data. General amino acid models are reflective of conservation at the protein level due to functional constraints, with most frequent amino acids exchanges taking place between residues with the same or similar physicochemical properties. In contrast...

‣ A single amino acid substitution that determines IEF band pattern of barley beta-amylase

Ma, Y.; Langridge, P.; Logue, S.; Evans, D.
Fonte: Academic Press Ltd Publicador: Academic Press Ltd
Tipo: Artigo de Revista Científica
Publicado em //2002 Português
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Barley beta -amylase exhibits two distinct band patterns after isoelectric focusing (IEF), termed Sd1 and Sd2. A comparison of the deduced amino acid sequences revealed five amino acid differences between the two types ofbeta -amylase. To investigate whether the two band patterns are due to these amino acid substitutions, four Sd2 mutants (Sd2-R115C, Sd2-D165E, Sd2-L347S and Sd2-V430A) were constructed by site-directed mutagenesis. The analysis of IEF band patterns of mutant and wild-type beta -amylases revealed that only the replacement of R115 with cysteine converted the Sd2 band pattern to the Sd1 one. The contribution of the R115C substitution to the IEF band pattern of barley beta -amylase was further confirmed by generating a Sd1 mutant (Sd1-C115R), where cysteine at this position was in turn replaced by arginine. As a result of this mutation, the Sd1 band pattern was converted into the Sd2 pattern. Calculation of the electrostatic potential and reducing agent treatment revealed that the R115C substitution altered both the net charge on the protein surface and intermolecular interactions by disulfide bonds, thereby altering the IEF band pattern of barley beta -amylase.; http://www.elsevier.com/wps/find/journaldescription.cws_home/622859/description#description

‣ Mapping of barley (Hordeum vulgare L.) beta-amylase alleles in which an amino acid substitution determines beta-amylase isoenzyme type and the level of free beta-amylase

Li, C.D.; Langridge, P.; Zhang, X.; Eckstein, P.; Rossnagel, B.; Lance, R.; Lefol, E.; Lu, M.; Harvey, B.; Scoles, G.
Fonte: Academic Press Ltd Elsevier Science Ltd Publicador: Academic Press Ltd Elsevier Science Ltd
Tipo: Artigo de Revista Científica
Publicado em //2002 Português
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The three beta -amylase genes (Bmy1, 2 and 3) in cultivated barley were mapped to chromosomes 4HL, 2HL And 4HL respectively using RFLP analysis. No recombinants between Bmy1 andBmy3 were detected among 264 DH lines. Polymorphism of the Sd1 and Sd2 isoenzymes of beta -amylase co-segregated with the Bmy loci on chromosome 4HL in a doubled-haploid population of the cross Chebec (Sd2)×Harrington (Sd1). This locus also explained 90·5% of the variation in the level of free enzyme between the two parents. Two cDNAs ofbeta -amylase were isolated by RT-PCR from the developing grains of Harrington (Sd1) and Galleon (Sd2). Alignment of the deduced amino acid sequences identified three amino-acid substitutions between the Sd2 and Sd1 forms of beta -amylase (Arg115 – Cys, Asp165 – Glu, and Val430 – Ala). Three allele-specific PCR primer pairs based on the three amino acid substitutions were used to amplify the beta -amylase genes in genomic DNA of sixteen barley cultivars/lines. Only the Arg115(Sd2)/Cys(Sd1) substitution was consistent with the isoenzyme form. This amino acid replacement reduced the pI of the Sd1 beta -amylase consistent with the fact that the Sd2 form is more basic than the Sd1 form when separated by IEF. The mutation from Arg115 to Cys in the Sd1 form also provides one more -SH group to form S-S-bridges. As bound beta -amylase is linked to the insoluble proteins of the endosperm and its inhibitor via disulphide bridges this could explain the higher level of binding exhibited by Sd1 vs Sd2. Thus a single amino acid substitution determines both the isoenzyme type and beta -amylase binding.; Li...

‣ Genome bias influences amino acid choices: analysis of amino acid substitution and re-compilation of substitution matrices exclusive to an AT-biased genome

Paila, Umadevi; Kondam, Rohini; Ranjan, Akash
Fonte: Oxford University Press Publicador: Oxford University Press
Tipo: Artigo de Revista Científica
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The genomic era has seen a remarkable increase in the number of genomes being sequenced and annotated. Nonetheless, annotation remains a serious challenge for compositionally biased genomes. For the preliminary annotation, popular nucleotide and protein comparison methods such as BLAST are widely employed. These methods make use of matrices to score alignments such as the amino acid substitution matrices. Since a nucleotide bias leads to an overall bias in the amino acid composition of proteins, it is possible that a genome with nucleotide bias may have introduced atypical amino acid substitutions in its proteome. Consequently, standard matrices fail to perform well in sequence analysis of these genomes. To address this issue, we examined the amino acid substitution in the AT-rich genome of Plasmodium falciparum, chosen as a reference and reconstituted a substitution matrix in the genome's context. The matrix was used to generate protein sequence alignments for the parasite proteins that improved across the functional regions. We attribute this to the consistency that may have been achieved amid the target and background frequencies calculated exclusively in our study. This study has important implications on annotation of proteins that are of experimental interest but give poor sequence alignments with standard conventional matrices.

‣ Amino acid similarity matrices based on force fields

Doszt�nyi, Zsuzsanna; Torda, Andrew
Fonte: Oxford University Press Publicador: Oxford University Press
Tipo: Artigo de Revista Científica
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Motivation: We propose a general method for deriving amino acid substitution matrices from low resolution force fields. Unlike current popular methods, the approach does not rely on evolutionary arguments or alignment of sequences or structures. Instead,

‣ Position Dependent and Independent Evolutionary Models Based on Empirical Amino Acid Substitution Matrices

Barbiellini, B.; Portnova, Alexandra; Chetoukhina, Anna; Lu, Chia-Hsin; Pellegrini, Matteo
Fonte: Universidade Cornell Publicador: Universidade Cornell
Tipo: Artigo de Revista Científica
Publicado em 19/05/2004 Português
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Evolutionary models measure the probability of amino acid substitutions occurring over different evolutionary distances. We examine various evolutionary models based on empirically derived amino acid substitution matrices. The models are constructed using the PAM and BLOSUM amino acid substitution matrices. We rescale these matrices by raising them to powers to model substitution patterns that account for different evolutionary distances. We also examine models that account for the dissimilarity of substitution rates along a protein sequence. We compare the models by computing the likelihood of each model across different alignments. We also present a specific example to illustrate the subtle differences in the estimation of evolutionary distance computed using the different models.; Comment: Paper presented at the Biological Language Conference November 20-21, 2003 University of Pittsburgh

‣ The effect of molecular shape on the thermotropic liquid crystal behavior of monolauroylated amino acid glyceride conjugates

Morán, María del Carmen; Pinazo Gassol, Aurora; Clapés Saborit, Pere; Infante, María Rosa; Pons Pons, Ramon
Fonte: American Chemical Society Publicador: American Chemical Society
Tipo: Artículo Formato: 22195 bytes; application/pdf
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10 pages, 6 figures, 5 tables.-- PMID: 16853983 [PubMed].-- Printed version published Nov 8, 2005.; Monoacylglycerol amino acid conjugates constitute a novel class of specific biocompatible surfactants that can be considered analogues to partial glycerides and lysophospholipids. They consist of one aliphatic chain and one polar head, i.e., the amino acid, linked through a glycerol moiety. In a previous work, we synthesized monolauroylated amino acid glyceride conjugates, 1-O-lauroyl-rac-glycero-3-O-(Nα-acetyl-l-amino acid), changing the amino acid headgroup systematically: arginine (compound 2), aspartic acid (compound 3), glutamic acid (compound 4), asparagine (compound 5), glutamine (compound 6), and tyrosine (compound 7), to elucidate the structure−properties relationship governing the occurrence of their polymorphism. The thermotropism of the new compounds was measured with polarizing light microscopy, differential scanning calorimetry, and X-ray diffraction and compared with the classical monoglyceride rac-1-lauroylglycerol (compound 1). The experiments were performed for a sequence of heating, cooling, and reheating scans. The results showed that compounds 1−6 exhibit a thermotropic smectic phase. As a consequence, the substitution of the polar head did not engender any curvature into the system...

‣ Functional effects of amino acid substitutions within the large binding pocket of the phosphotriesterase OpdA from Agrobacterium sp. P230

Horne, Irene; Qiu, Xinghui; Ollis, David; Russell, Robyn; Oakeshott, John Graham
Fonte: Elsevier Publicador: Elsevier
Tipo: Artigo de Revista Científica
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The phosphotriesterase OpdA from Agrobacterium sp. P230 has about 10-fold higher activity for dimethyl organophosphate (OP) insecticides, than its homologue from Flavobacterium sp. ATCC27551, organophosphate hydrolase (OPH). OpdA shows about 10% amino acid sequence divergence from OPH and also has a 20 residue C-terminal extension. Here we show that the difference in kinetics is largely explained by just two amino acid differences between the two proteins. A truncated form of OpdA demonstrated that the C-terminal extension has no effect on its preference for dimethyl organophosphate substrates. Chimeric proteins of OPH and OpdA were then analysed to show that replacement of a central region of OpdA sequence, which encodes the residues in the large subsite of the active site, with the homologous region in OPH decreased the activity of OpdA towards dimethyl OPs, to values close to those for OPH. Site-directed mutagenesis in this region identified two differences between the proteins, Y257H and F272L (with the OpdA residues first) as being responsible for this reduction. These two differences were also responsible for the increased activity of OpdA towards the diisopropyl organophosphate, diisopropyl fluorophosphate, relative to OPH. Molecular modelling of triethyl phosphate in the active site of OpdA confirmed a reduction in the size of the large subsite relative to OPH.

‣ The same amino acid substitution in orthologous esterases confers organophosphate resistance on the house fly and a blowfly

Claudianos, Charles; Russell, Robyn; Oakeshott, John Graham
Fonte: Pergamon-Elsevier Ltd Publicador: Pergamon-Elsevier Ltd
Tipo: Artigo de Revista Científica
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Organophosphate (OP) insecticide resistance in certain strains of Musca domestica is associated with reduction in the carboxylesterase activity of a particular esterase isozyme. This has been attributed to a 'mutant ali- esterase hypothesis', which invokes a structural mutation to an ali-esterase resulting in the loss of its carboxylesterase activity but acquisition of OP hydrolase activity. It has been shown that the mutation in Lucilia cuprina is a Gly137 → Asp substitution in the active site of an esterase encoded by the LcαE7 gene (Newcomb, R.D., Campbell, P.M., Ollis, D.L., Cheah, E., Russell, R.J., Oakeshott, J.G., 1997. A single amino acid substitution converts a carboxylesterase to an organophosphate hydrolase and confers insecticide resistance on a blowfly. Proc. Natl. Acad. Sci. USA 94, 7464- 7468). We now report the cloning and characterisation of the orthologous M. domestica MdαE7 gene, including the sequencing of cDNAs from the OP resistant Rutgers and OP susceptible sbo and WHO strains. The MdαE7 gene has the same intron structure as LcαE7 and encodes a protein with 76% amino acid identity to LcαE7. Comparisons between susceptible and resistance alleles show resistance in M. domestica is associated with the same Gly137 → Asp mutation as in L. cuprina. Bacterial expression of the Rutgers allele shows its product has OP hydrolase activity. The data indicate identical catalytic mechanisms have evolved in orthologous MdαE7 and LcαE7 molecules to endow diazinon-type resistance on the two species of higher Diptera.