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Novel Activity (novel + activity)
Selected AbstractsA ,-1,4-Galactosyltransferase from Helicobacter pylori is an Efficient and Versatile Biocatalyst Displaying a Novel Activity for Thioglycoside SynthesisCHEMBIOCHEM, Issue 10 2008Darius-Jean Namdjou Abstract Helicobacter pylori is a highly persistent and common pathogen in humans. It is the causative agent of chronic gastritis and its further stages. HP0826 is the ,-1,4-galactosyltransferase involved in the biosynthesis of the LPS O-chain backbone of H. pylori. Though it was first cloned nearly a decade ago, there are surprisingly limited data about the characteristics of HP0826, especially given its prominent role in H. pylori pathogenicity. We here demonstrate that HP0826 is a highly efficient and promiscuous biocatalyst. We have exploited two novel enzymatic activities for the quantitative synthesis of the thiodisaccharide Gal-,- S -1,4-GlcNAc- pNP as well as Gal-,-1,4-Man- pNP. We further show that Neisseria meningitidis ,-1,4-galactosyltransferases LgtB can be used as an equally efficient catalyst in the latter reaction. Thiodisaccharides have been extensively used in structural biology but can also have therapeutic uses. The Gal-,-1,4-Man linkage is found in the Leishmania species LPG backbone disaccharide repeats and cap, which have been associated with vector binding in Leishmaniasis. [source] Studies into factors contributing to substrate specificity of membrane-bound 3-ketoacyl-CoA synthasesFEBS JOURNAL, Issue 19 2002Brenda J. Blacklock We are interested in constructing a model for the substrate-binding site of fatty acid elongase-1 3-ketoacyl CoA synthase (FAE1 KCS), the enzyme responsible for production of very long chain fatty acids of plant seed oils. Arabidopsis thaliana and Brassica napus FAE1 KCS enzymes are highly homologous but the seed oil content of these plants suggests that their substrate specificities differ with respect to acyl chain length. We used in vivo and in vitro assays of Saccharomyces cerevisiae -expressed FAE1 KCSs to demonstrate that the B. napus FAE1 KCS enzyme favors longer chain acyl substrates than the A. thaliana enzyme. Domains/residues responsible for substrate specificity were investigated by determining catalytic activity and substrate specificity of chimeric enzymes of A. thaliana and B. napus FAE1 KCS. The N-terminal region, excluding the transmembrane domain, was shown to be involved in substrate specificity. One chimeric enzyme that included A. thaliana sequence from the N terminus to residue 114 and B. napus sequence from residue 115 to the C terminus had substrate specificity similar to that of A. thaliana FAE1 KCS. However, a K92R substitution in this chimeric enzyme changed the specificity to that of the B. napus enzyme without loss of catalytic activity. Thus, this study was successful in identifying a domain involved in determining substrate specificity in FAE1 KCS and in engineering an enzyme with novel activity. [source] Promiscuous Acylases-Catalyzed Markovnikov Addition of N-Heterocycles to Vinyl Esters in Organic MediaADVANCED SYNTHESIS & CATALYSIS (PREVIOUSLY: JOURNAL FUER PRAKTISCHE CHEMIE), Issue 4-5 2006Wei-Bo Wu Abstract Three acylases, including D -aminoacylase from Escherichia coli, acylase "Amano" from Aspergillus oryzae and immobilized penicillin G acylase from Escherichia coli have been found to possess novel activity to catalyze the Markovnikov addition reaction of N-heterocycles to vinyl esters. The aza-Markovnikov addition reactions of 4-nitroimidazle to vinyl acetate catalyzed by D -aminoacylase, acylase "Amano" and immobilized penicillin G acylase were up to 1260-fold, 720-fold and 320-fold faster than the respective non-enzymatic reaction. Some control experiments have been designed to demonstrate the catalytic specificity of acylases. Under the catalysis of these promiscuous acylases, a number of N-heterocycles, including some pentacyclic N-heterocycles, pyrimidines and purines, were successfully added to a series of vinyl esters in moderate to excellent yields to prepare N-heterocycle derivatives. The acylase-catalyzed Markovnikov addition reaction has provided a new strategy to perform the Markovnikov addition and expanded the application of biocatalysts. [source] Thrombin and PAR-1 stimulate differentiation of bone marrow-derived endothelial progenitor cellsJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 3 2006S. T. TARZAMI Summary., Endothelial progenitor cells (EPCs) from the bone marrow play an important role in vascular response to injury and ischemia. The mediators involved in the mobilization, recruitment, proliferation and differentiation of EPCs are not fully understood. In this study, the role of coagulation factor thrombin and protease-activated receptor-1 (PAR-1) on bone marrow-derived cell proliferation and differentiation was investigated. Bone marrow cells (BMCs) were isolated from C57/BL6 mice and plated on fibronectin-coated flasks. Cell characteristics, proliferation and the expression of endothelial cell markers were determined using immunohistochemistry, thymidine uptake and fluorescence activated-cell sorting (FACS), respectively. The results show that thrombin stimulated enrichment of bone marrow cells with endothelial morphology, exhibiting acetylated-low-density lipoprotein (LDL) uptake and isolectin staining. Thrombin or PAR-1-activating peptide produced a 2- to 3-fold increase in the total number of cells as well as an increase in vascular endothelial (VE)-cadherin-positive cells. Thrombin treatment of VE-cadherin-negative cells prepared after cell sorting resulted in the generation of 3- to 4-fold higher VE-cadherin-positive cells than the untreated cultures. Increase in VE-cadherin-positive cells was inhibited by hirudin and efegatran. These results provide first evidence for a novel activity of thrombin and PAR-1 on bone marrow progenitor cell proliferation and EPC differentiation, and suggest their potential role in vascular regeneration and recanalization of thrombus. [source] Ultraviolet B Radiation of Human Skin Generates Platelet-activating Factor Receptor AgonistsPHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 4 2010Jared B. Travers Ultraviolet B radiation (UVB) is a potent stimulator of epidermal cytokine production. In addition to cytokines, such as tumor necrosis factor-alpha (TNF-,), UVB generates bioactive lipids including platelet-activating factor (PAF). Our previous in vitro studies in keratinocytes or epithelial cell lines have demonstrated that UVB-mediated production of PAF agonists is due primarily to the pro-oxidative effects of this stimulant, resulting in the nonenzymatic production of modified phosphocholines (oxidized glycerophosphocholines). The current studies use human skin to assess whether UVB irradiation generates PAF-receptor agonists, and the role of oxidative stress in their production. These studies demonstrate that UVB irradiation of human skin results in PAF agonists, which are blocked by the antioxidant vitamin C and the epidermal growth factor receptor inhibitor PD168393. Inasmuch as UVB-generated PAF agonists have been implicated in animal model systems as being involved in photobiologic processes including systemic immunosuppression and cytokine (TNF-,) production, these studies indicate that this novel activity could be involved in human disease. [source] Insights into drug metabolism by cytochromes P450 from modelling studies of CYP2D6-drug interactionsBRITISH JOURNAL OF PHARMACOLOGY, Issue S1 2008J-D Maréchal The cytochromes P450 (CYPs) comprise a vast superfamily of enzymes found in virtually all life forms. In mammals, xenobiotic metabolizing CYPs provide crucial protection from the effects of exposure to a wide variety of chemicals, including environmental toxins and therapeutic drugs. Ideally, the information on the possible metabolism by CYPs required during drug development would be obtained from crystal structures of all the CYPs of interest. For some years only crystal structures of distantly related bacterial CYPs were available and homology modelling techniques were used to bridge the gap and produce structural models of human CYPs, and thereby obtain useful functional information. A significant step forward in the reliability of these models came seven years ago with the first crystal structure of a mammalian CYP, rabbit CYP2C5, followed by the structures of six human enzymes, CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2D6 and CYP3A4, and a second rabbit enzyme, CYP2B4. In this review we describe as a case study the evolution of a CYP2D6 model, leading to the validation of the model as an in silico tool for predicting binding and metabolism. This work has led directly to the successful design of CYP2D6 mutants with novel activity,including creating a testosterone hydroxylase, converting quinidine from inhibitor to substrate, creating a diclofenac hydroxylase and creating a dextromethorphan O -demethylase. Our modelling-derived hypothesis-driven integrated interdisciplinary studies have given key insight into the molecular determinants of CYP2D6 and other important drug metabolizing enzymes. British Journal of Pharmacology (2008) 153, S82,S89; doi:10.1038/sj.bjp.0707570; published online 19 November 2007 [source] | |||||||||||||