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Substrate Spectrum (substrate + spectrum)
Selected AbstractsScreening, Characterization and Application of Cyanide-resistant Nitrile HydratasesENGINEERING IN LIFE SCIENCES (ELECTRONIC), Issue 6 2004T. Gerasimova Abstract Two new bacterial strains, Pseudomonas marginales MA32 and Pseudomonas putida MA113, containing nitrile hydratases resistant to cyanide were isolated from soil samples by an enrichment procedure. In contrast to known nitrile hydratases, which rapidly lose activity at low to moderate cyanide concentrations, the enzymes described in this paper tolerate up to 50 mM cyanide. They show a broad substrate spectrum including not only small substrates like acrylonitrile but also nitriles with longer side chains and even nitriles with quarternary alpha-carbon atoms. Both characteristics are essential for the transformation of ketone cyanohydrins, which are much more instable and therefore releasing much higher amounts of prussic acid than cyanohydrins formed from aldehydes. P. marginales MA32 was used as a whole cell biocatalyst for the hydration of acetone cyanohydrin to ,-Hydroxyisobutyramide, which is a precursor of methacrylamide, an important pre-polymer. After optimization of the process conditions a maximum amide concentration of more than 1.6 M could be reached within 5 hours with 5,g/L biocatalyst referred to cell dry weight. [source] High resolution structure and catalysis of O -acetylserine sulfhydrylase isozyme B from Escherichia coliFEBS JOURNAL, Issue 20 2007Georg Zocher The crystal structure of the dimeric O -acetylserine sulfhydrylase isozyme B from Escherichia coli (CysM), complexed with the substrate analog citrate, has been determined at 1.33 Å resolution by X-ray diffraction analysis. The C1-carboxylate of citrate was bound at the carboxylate position of O -acetylserine, whereas the C6-carboxylate adopted two conformations. The activity of the enzyme and of several active center mutants was determined using an assay based on O -acetylserine and thio-nitrobenzoate (TNB). The unnatural substrate TNB was modeled into the reported structure. The substrate model and the observed mutant activities may facilitate future protein engineering attempts designed to broaden the substrate spectrum of the enzyme. A comparison of the reported structure with previously published CysM structures revealed large conformational changes. One of the crystal forms contained two dimers, each of which comprised one subunit in a closed and one in an open conformation. Although the homodimer asymmetry was most probably caused by crystal packing, it indicates that the enzyme can adopt such a state in solution, which may be relevant for the catalytic reaction. [source] 17, -Hydroxysteroid dehydrogenase type 11 is a major peroxisome proliferator-activated receptor ,-regulated gene in mouse intestineFEBS JOURNAL, Issue 20 2004Kiyoto Motojima In order to study the role of peroxisome proliferator-activated receptor , in mouse intestine, its agonist-induced proteins were identified by peptide mass fingerprinting followed by Northern blot analysis using their cDNAs. One of the most remarkably induced proteins was identified as 17,-hydroxysterol dehydrogenase type 11. Its very rapid induction by various agonists was most efficient in intestine and then in liver. These findings together with recently reported results showing the enzyme family's wide substrate spectrum, including not only glucocorticoids and sex steroids but also bile acids, fatty acids and branched chain amino acids, suggest new roles for both peroxisome proliferator-activated receptor , and 17,-hydroxysterol dehydrogenase type 11 in lipid metabolism and/or detoxification in the intestine. [source] Cutting Long Syntheses Short: Access to Non-Natural Tyrosine Derivatives Employing an Engineered Tyrosine Phenol LyaseADVANCED SYNTHESIS & CATALYSIS (PREVIOUSLY: JOURNAL FUER PRAKTISCHE CHEMIE), Issue 4 2010Birgit Seisser Abstract The chemical synthesis of 3-substituted tyrosine derivatives requires a minimum of four steps to access optically enriched material starting from commercial precursors. Attempting to short-cut the cumbersome chemical synthesis of 3-substituted tyrosine derivatives, a single step biocatalytic approach was identified employing the tyrosine phenol lyase from Citrobacter freundii. The enzyme catalyses the hydrolysis of tyrosine to phenol, pyruvate and ammonium as well as the reverse reaction, thus the formation of tyrosine from phenol, pyruvate and ammonium. Since the wild-type enzyme possessed a very narrow substrate spectrum, structure-guided, site-directed mutagenesis was required to change the substrate specificity of this CC bond forming enzyme. The best variant M379V transformed, for example, o -cresol, o -methoxyphenol and o -chlorophenol efficiently to the corresponding tyrosine derivatives without any detectable side-product. In contrast, all three phenol compounds were non-substrates for the wild-type enzyme. Employing the mutant, various L -tyrosine derivatives (3-Me, 3-OMe, 3-F, 3-Cl) were obtained with complete conversion and excellent enantiomeric excess (>97%) in just a single ,green' step starting from pyruvate and commercially available phenol derivatives. [source] Identification of potential substrate proteins for the periplasmic Escherichia coli chaperone SkpPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 23-24 2008Svenja Jarchow Abstract The "seventeen kilodalton protein" (Skp) is a predominant periplasmic chaperone of Escherichia coli, which is involved in the biogenesis of abundant outer membrane proteins (OMPs) such as OmpA, PhoE, and LamB. In this study the substrate profile of Skp was investigated in a proteomics approach. Skp was overexpressed in a deficient E. coli strain as a fusion protein with the Strep,tag and captured, together with any host proteins associated with it, from the periplasmic cell extract under mild conditions via one-step Strep,Tactin affinity chromatography. Copurified substrate proteins were then identified by high resolution 2-DE with immobilized pH-gradients, followed by MALDI-TOF MS. Apart from the known Skp substrates, including OmpA and LamB, more than 30 other interacting proteins were detected, especially from the outer membrane, among these FadL and BtuB, and from the periplasm such as MalE and OppA. Thus, Skp does not only serve as a specialized chaperone for a small set of OMPs, but it seems to exhibit a broader substrate spectrum, including soluble periplasmic proteins. These findings should prompt further investigation into the physiological role of Skp and may promote its use for the bacterial production of biochemically active heterologous proteins whose folding requires secretion into the oxidizing milieu of the periplasm. [source] Male gametophyte development in bread wheat (Triticum aestivum L.): molecular, cellular, and biochemical analyses of a sporophytic contribution to pollen wall ontogenyTHE PLANT JOURNAL, Issue 6 2002Aiming Wang Summary Bread wheat (hexaploid AABBDD genome; 16 billion basepairs) is a genetically complex, self-pollinating plant with bisexual flowers that produce short-lived pollen. Very little is known about the molecular biology of its gametophyte development despite a longstanding interest in hybrid seeds. We present here a comprehensive characterization of three apparently homeologous genes (TAA1a, TAA1b and TAA1c) and demonstrate their anther-specific biochemical function. These eight-exon genes, found at only one copy per haploid complement in this large genome, express specifically within the sporophytic tapetum cells. The presence of TAA1 mRNA and protein was evident only at specific stages of pollen development as the microspore wall thickened during the progression of free microspores into vacuolated-microspores. This temporal regulation matched the assembly of wall-impregnated sporopollenin, a phenylpropanoid-lipid polymer containing very long chain fatty alcohols (VLCFAlc), described in the literature. Our results establish that sporophytic genes contribute to the production of fatty alcohols: Transgenic expression of TAA1 afforded production of long/VLCFAlc in tobacco seeds (18 : 1; 20 : 1; 22 : 1; 24 : 0; 26 : 0) and in Escherichia coli (14 : 0; 16 : 0; 18 : 1), suggesting biochemical versatility of TAA1 with respect to cellular milieu and substrate spectrum. Pollen walls additionally contain fatty alcohols in the form of wax esters and other lipids, and some of these lipids are known to play a role in the highly specific sexual interactions at the pollen,pistil interface. This study provides a handle to study these and to manipulate pollen traits, and, furthermore, to understand the molecular biology of fatty alcohol metabolism in general. [source] Crystallization and preliminary X-ray crystallographic analyses of CMY-1 and CMY-10, plasmidic class C ,-lactamases with extended substrate spectrumACTA CRYSTALLOGRAPHICA SECTION D, Issue 2 2004Sun-Joo Lee Plasmid-encoded class C ,-lactamases, including CMY-1 and CMY-10, hydrolyze the lactam bonds of ,-lactam antibiotics, inducing therapeutic failure and a lack of eradication of clinical isolates by third-generation cephalosporins or cephamycins. Therefore, the enzymes are potential targets for developing agents against pathogens isolated from patients suffering from wound infection, urinary tract infection or pneumonia. CMY-1 and CMY-10 were purified and crystallized at 298,K. X-ray diffraction data from CMY-1 and CMY-10 crystals have been collected to 2.5 and 1.5,Å resolution, respectively, using synchrotron radiation. The crystals of the two proteins are isomorphous and belong to the primitive monoclinic space group P21. [source] Insights into Sequence,Activity Relationships amongst Baeyer,Villiger Monooxygenases as Revealed by the Intragenomic Complement of Enzymes from Rhodococcus jostii RHA1CHEMBIOCHEM, Issue 7 2009Claudia Szolkowy Abstract TheRhodococcus jostiiRHA1 genome encodes a number of enzymes that can be exploited as biocatalysts. Study of the substrate spectrum and enantioselectivity of Baeyer,Villiger monooxygenases from R. jostii allowed the identification of short amino acid sequences specific to groups displaying certain catalytic characteristics. The gel illustrates the substrate acceptance spectra and selectivities of the different proteins. Microbial genome sequences are providing a wealth of information on new enzymes that have considerable potential as biocatalysts. The recently sequenced genome of Rhodococcus jostii RHA1, for example, has revealed an impressive array of catabolic enzymes, including many putative Baeyer,Villiger monooxygenases (BVMOs). We have cloned 23 target BVMO sequences from the genome of R. jostii RHA1 and heterologously expressed 13 of these as soluble proteins to unearth new substrate specificities and selectivities. Whole-cell biocatalysts expressing the genes were screened against seven different test substrates. Each of these catalysts displayed activity toward at least three ketones. We observed a remarkable diversity of both regio- and enantioselectivity among the BVMOs from R. jostii RHA1 for the transformation of two chiral substrates, with some enzymes displaying high enantioselectivity for the isomers of 2-methylcyclopentanone. With the notable exception of the product of gene ro03437, named MO14, the biocatalysts' sequences correlated well with their respective activities and selectivities. This correlation allowed the identification of sequence motifs specific to subgroups of the BVMOs from R. jostii and other organisms. Overall, the data improve predictive models of BVMO activity from sequence and suggest new avenues to pursue in engineering these enzymes. [source] | |||||||||||||