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Amino Acid Exchanges (amino + acid_exchanges)
Selected AbstractsSequence diversity of the peptaibol antibiotic suzukacillin-A from the mold Trichoderma virideJOURNAL OF PEPTIDE SCIENCE, Issue 5 2006Corina Krause Abstract From the culture broth of the mold Trichoderma viride, strain 63 C-I, the polypeptide antibiotic suzukacillin (SZ) was isolated. A peptide mixture named SZ-A was obtained by crystallization from crude SZ. Individual peptides from SZ-A were isolated by semipreparative HPLC and sequences were determined by HPLC-ESI-MS. The data confirm a general sequence of SZ-A published previously and in addition establish the individual sequences of 15 acetylated eicosa peptides with C -terminal alcohols. The major peptide SZ-A4 (21% of all peptides) shows the sequence: Ac-Aib-Ala-Aib-Ala-Aib-Ala6 -Gln-Aib-Lx9 -Aib-Gly-Aib12 -Aib-Pro-Vx15 -Aib-Vx17 -Gln-Gln-Fol. Amino acid exchanges of the peptaibol are located in position 6 (Ala/Aib), 9 (Vx/Lx), 12 (Aib/Lx), 17 (Aib/Vx) and possibly at position15 (Val/Iva) (uncommon abbreviations: Aib (,-aminoisobutyric acid); Iva (D -isovaline); Lx (L -leucine or L -isoleucine); Vx (L -valine or D -isovaline); Fol (L -phenylalaninol)). Copyright © 2005 European Peptide Society and John Wiley & Sons, Ltd. [source] The metabolic role and evolution of l -arabinitol 4-dehydrogenase of Hypocrea jecorinaFEBS JOURNAL, Issue 10 2004Manuela Pail l -Arabinitol 4-dehydrogenase (Lad1) of the cellulolytic and hemicellulolytic fungus Hypocrea jecorina (anamorph: Trichoderma reesei) has been implicated in the catabolism of l -arabinose, and genetic evidence also shows that it is involved in the catabolism of d -xylose in xylitol dehydrogenase (xdh1) mutants and of d -galactose in galactokinase (gal1) mutants of H. jecorina. In order to identify the substrate specificity of Lad1, we have recombinantly produced the enzyme in Escherichia coli and purified it to physical homogeneity. The resulting enzyme preparation catalyzed the oxidation of pentitols (l -arabinitol) and hexitols (d -allitol, d -sorbitol, l -iditol, l -mannitol) to the same corresponding ketoses as mammalian sorbitol dehydrogenase (SDH), albeit with different catalytic efficacies, showing highest kcat/Km for l -arabinitol. However, it oxidized galactitol and d -talitol at C4 exclusively, yielding l -xylo-3-hexulose and d -arabino-3-hexulose, respectively. Phylogenetic analysis of Lad1 showed that it is a member of a terminal clade of putative fungal arabinitol dehydrogenase orthologues which separated during evolution of SDHs. Juxtapositioning of the Lad1 3D structure over that of SDH revealed major amino acid exchanges at topologies flanking the binding pocket for d -sorbitol. A lad1 gene disruptant was almost unable to grow on l -arabinose, grew extremely weakly on l -arabinitol, d -talitol and galactitol, showed reduced growth on d -sorbitol and d -galactose and a slightly reduced growth on d -glucose. The weak growth on l -arabinitol was completely eliminated in a mutant in which the xdh1 gene had also been disrupted. These data show not only that Lad1 is indeed essential for the catabolism of l -arabinose, but also that it constitutes an essential step in the catabolism of several hexoses; this emphasizes the importance of such reductive pathways of catabolism in fungi. [source] Mutations towards enantioselectivity adversely affect secretion of Pseudomonas aeruginosa lipaseFEMS MICROBIOLOGY LETTERS, Issue 1 2008Sascha Hausmann Abstract Lipases are important biocatalysts used as detergent additives to manufacture biodiesel, and in particular, for the production of enantiopure compounds such as alcohols, amines and carboxylic acids. Extensive efforts were conducted trying to optimize lipase properties and lipase LipA of Pseudomonas aeruginosa comprises the best-studied example in terms of optimizing enantioselectivity by application of numerous directed evolution methods. Its enantioselectivity in the asymmetric hydrolysis of the model substrate 2-methyldecanoic acid p -nitrophenyl ester was increased from E=1.1 for the wild-type enzyme to E=51 for the best (S)-enantioselective variant which carried six amino acid exchanges. We have observed that overexpression of this variant in the homologous host resulted in only marginal yields of enzyme in the bacterial culture supernatant, suggesting that the enantioselective LipA variant was secreted with only low efficiency. Hence, we have analysed the secretion of this lipase variant and compared it to variants carrying either the respective single mutations or some combinations. We report here the identification of two amino acid substitutions located on the protein surface, which significantly impair lipase secretion. [source] Factor IX mutants with enhanced catalytic activityJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 10 2009R. HARTMANN Summary.,Background:,Activated coagulation factor IX (FIXa) has low catalytic activity towards its physiologic substrate FX when activated FVIII (FVIIIa) is absent. One reason for this is that the FIX surface loop 99 stabilizes FIXa in a conformation that limits access of FX to the active site. Objectives:,To investigate the effect of mutations in loop 99 and in the active site on FIXa activity with and without FVIIIa. Methods:,Five full-length FIX mutants with amino acid exchanges in the catalytic domain of FIX were constructed and characterized by measuring their activity in FX activation in model systems and in plasma. Results and Conclusions:,The mutants showed no or marginally improved catalytic properties in FX activation by the intrinsic tenase complex (FIXa,FVIIIa,Ca2+,phospholipid). The combination of mutations Y94F and K98T hardly affected FX activation in the presence of FVIIIa, but yielded a FIX molecule that, in FIX-depleted plasma, had , 2.5-fold higher clotting activity and , 3.5-fold higher activity in a thrombin generation assay than plasma-derived FIX (pdFIX). Two FIXa mutants had considerably increased activities towards FX in the absence of FVIIIa. FIXa-Y94F/K98T/Y177F/I213V/E219G (FIXa-L) and FIXa-Y94F/A95aK/K98T/Y177F/I213V/E219G (FIXa-M) activated FX with catalytic efficiencies (kcat/Km) that, as compared with activated pdFIX, were increased 17-fold and six-fold, respectively. However, in plasma, their zymogen forms performed similarly to pdFIX. This indicates that the introduced mutations not only affected the activity of FIXa but may have also influenced the lifetime of the activated mutant molecules in plasma by modifying their activation and/or inhibition rates. [source] Identification and molecular analysis of candidate genes homologous to HcrVf genes for scab resistance in applePLANT BREEDING, Issue 1 2009A. Boudichevskaia Abstract The genetic locus for resistance to apple scab most frequently used in apple breeding is Vf, derived from Malus floribunda 821. For the Vf locus a cluster of four resistance gene paralogs (called as HcrVf genes) encoding receptor-like proteins (RLP) with similarity to the tomato Cf resistance genes is known. Based on published sequences for HcrVf1 and HcrVf2 PCR primers were designed from the domain B and the variable leucine-rich repeat (LRR) C1 subdomain. PCR products with high amino acid identity (85,100%) to HcrVf1 and HcrVf2 were obtained not only from M. floribunda 821 and Vf cultivars but also from other apple scab resistance sources, such as ,Russian Seedling' R12740-7A (Vr resistance) or ,Antonovka polutorafuntovaya' (VA resistance). A series of 13 HcrVf candidate genes have been partly cloned from the PCR fragments spanning N-terminal LRRs 20,30. A considerable number of amino acid exchanges within the solvent-exposed xxLxLxx structural motives were detected among the homologous sequences. Expression analyses and mapping focused on a selected Vf- homologous candidate gene (called Vf2ARD) identified in resistant Malus genotypes known for carrying other scab resistance genes than Vf. RT-PCR experiments showed that Vf2ARD is expressed under pathogen-free conditions. The results of a quantitative PCR-based transcription profiling suggest that this gene is scab-inducible in some resistant cultivars. Vf2ARD has been mapped on linkage group LG 1. It is separated from the Vf gene cluster with a genetic distance of about 2 cM and might be a member of a second Vf - like locus on apple linkage group LG 1. [source] Characterization of the porcine transferrin gene (TF,) and its association with disease severity following an experimental Actinobacillus pleuropneumoniae infectionANIMAL GENETICS, Issue 4 2010E. Dani, owicz Summary Transferrin (TF)-mediated provision of iron is essential for a productive infection by many bacterial pathogens, and iron-depletion of TF is a first line defence against bacterial infections. Therefore, the transferrin (TF) gene can be considered a candidate gene for disease resistance. We obtained the complete DNA sequence of the porcine TF gene, which spans 40 kb and contains 17 exons. We identified polymorphisms on a panel of 10 different pig breeds. Comparative intra- and interbreed sequence analysis revealed 62 polymorphisms in the TF gene including one microsatellite. Ten polymorphisms were located in the coding sequence of the TF gene. Four SNPs (c.902A>T, c.980G>A, c.1417A>G, c.1810A>C) were predicted to cause amino acid exchanges (p.Lys301Ile, p.Arg327Lys, p.Lys473Glu, p.Asn604His). We performed association analyses using six selected TF markers and 116 pigs experimentally infected with Actinobacillus pleuropneumoniae serotype 7. The analysis showed breed-specific TF allele frequencies. In German Landrace, we found evidence for a possible association of the severity of A. pleuropneumoniae infection with TF genotypes. [source] | |||||||||||||