Home About us Contact
|
Home About us Contact | ||
|
|
||
De Novo Biosynthesis (de + novo_biosynthesis)
Selected AbstractsAcetyl-CoA:1- O -alkyl- sn -glycero-3-phosphocholine acetyltransferase (lyso-PAF AT) activity in cortical and medullary human renal tissueFEBS JOURNAL, Issue 14 2003Tzortzis N Nomikos Platelet-activating factor (PAF) is one of the most potent inflammatory mediators. It is biosynthesized by either the de novo biosynthesis of glyceryl ether lipids or by remodeling of membrane phospholipids. PAF is synthesized and catabolized by various renal cells and tissues and exerts a wide range of biological activities on renal tissue suggesting a potential role during renal injury. The aim of this study was to identify whether cortex and medulla of human kidney contain the acetyl-CoA:1- O -alkyl- sn -glycero-3-phosphocholine acetyltransferase (lyso-PAF AT) activity which catalyses the last step of the remodeling biosynthetic route of PAF and is activated in inflammatory conditions. Cortex and medulla were obtained from nephrectomized patients with adenocarcinoma and the enzymatic activity was determined by a trichloroacetic acid precipitation method. Lyso-PAF AT activity was detected in both cortex and medulla and distributed among the membrane subcellular fractions. No statistical differences between the specific activity of cortical and medullary lyso-PAF AT was found. Both cortical and medullary microsomal lyso-PAF ATs share similar biochemical properties indicating common cellular sources. [source] The essential neutral sphingomyelinase is involved in the trafficking of the variant surface glycoprotein in the bloodstream form of Trypanosoma bruceiMOLECULAR MICROBIOLOGY, Issue 6 2010Simon A. Young Summary Sphingomyelin is the main sphingolipid in Trypanosoma brucei, the causative agent of African sleeping sickness. In vitro and in vivo characterization of the T. brucei neutral sphingomyelinase demonstrates that it is directly involved in sphingomyelin catabolism. Gene knockout studies in the bloodstream form of the parasite indicate that the neutral sphingomyelinase is essential for growth and survival, thus highlighting that the de novo biosynthesis of ceramide is unable to compensate for the loss of sphingomyelin catabolism. The phenotype of the conditional knockout has given new insights into the highly active endocytic and exocytic pathways in the bloodstream form of T. brucei. Hence, the formation of ceramide in the endoplasmic reticulum affects post-Golgi sorting and rate of deposition of newly synthesized GPI-anchored variant surface glycoprotein on the cell surface. This directly influences the corresponding rate of endocytosis, via the recycling endosomes, of pre-existing cell surface variant surface glycoprotein. The trypanosomes use this coupled endocytic and exocytic mechanism to maintain the cell density of its crucial variant surface glycoprotein protective coat. TbnSMase is therefore genetically validated as a drug target against African trypanosomes, and suggests that interfering with the endocytic transport of variant surface glycoprotein is a highly desirable strategy for drug development against African trypanosomasis. [source] Structural studies of MIP synthaseACTA CRYSTALLOGRAPHICA SECTION D, Issue 3 2000Adam J. Stein The conversion of glucose 6-phosphate to 1- l - myo -inositol 1-phosphate (MIP) by 1- l - myo -inositol 1-phosphate synthase (MIP synthase) is the first committed and rate-limiting step in the de novo biosynthesis of inositol in all eukaryotes. The importance of inositol-containing molecules both as membrane components and as critical second messenger signal-transduction species make the function and regulation of this enzyme important for a host of biologically important cellular functions including proliferation, neurostimulation, secretion and contraction. MIP synthase has been overexpressed in Esherichia coli and purified to homogeneity by chromatographic methods. Two crystal forms of MIP synthase were obtained by the hanging-drop vapor-diffusion method. Native data sets for both crystal forms were collected in-house on a Rigaku R-AXIS IIC imaging-plate detector. Crystal form I belongs to space group C2, with unit-cell parameters a = 153.0, b = 96.6, c = 122.6,Å, , = 126.4°, and diffracts to 2.5,Å resolution. Crystal form II belongs to space group P21, with unit-cell parameters a = 94.5, b = 186.2, c = 86.5,Å, , = 110.5°, and diffracts to 2.9,Å resolution. [source] Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of SAICAR synthase from Streptococcus suis serotype 2ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 8 2010Xia Cheng Phosphoribosylaminoimidazole-succinocarboxamide synthase (SAICAR synthase) plays an essential role in the de novo biosynthesis of purine nucleotides. In this study, the SAICAR synthase from Streptococcus suis was cloned and overexpressed in Escherichia coli. The subsequent product was purified and crystallized using the hanging-drop vapour-diffusion method. The crystals diffracted to 2.8,Å resolution and belonged to space group P2, with unit-cell parameters a = 70.2, b = 52.2, c = 153.9,Å, , = 102.8°. [source] Purification, crystallization and preliminary X-ray analysis of the aspartate aminotransferase of Plasmodium falciparumACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 4 2010Rishabh Jain Aspartate aminotransferases (EC 2.6.1.1) catalyse the conversion of aspartate and ,-ketoglutarate to oxaloacetate and glutamate in a reversible manner. Thus, the aspartate aminotransferase of Plasmodium falciparum (PfAspAT) plays a central role in the transamination of amino acids. Recent findings suggest that PfAspAT may also play a pivotal role in energy metabolism and the de novo biosynthesis of pyrimidines. While therapeutics based upon the inhibition of other proteins in these pathways are already used in the treatment of malaria, the advent of multidrug-resistant strains has limited their efficacy. The presence of PfAspAT in these pathways may offer additional opportunities for the development of novel therapeutics. In order to gain a deeper understanding of the function and role of PfAspAT, it has been expressed and purified to homogeneity. The successful crystallization of PfAspAT, the collection of a 2.8,Å diffraction data set and initial attempts to solve the structure using molecular replacement are reported. [source] Cloning, expression, purification, crystallization and preliminary X-ray crystallographic study of the putative SAICAR synthetase (PH0239) from Pyrococcus horikoshii OT3ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 2 2010Kavyashree Manjunath The study of proteins involved in de novo biosynthesis of purine nucleotides is central in the development of antibiotics and anticancer drugs. In view of this, a protein from the hyperthermophile Pyrococcus horikoshii OT3 was isolated, purified and crystallized using the microbatch method. Its primary structure was found to be similar to that of SAICAR synthetase, which catalyses the seventh step of de novo purine biosynthesis. A diffraction-quality crystal was obtained using Hampton Research Crystal Screen II condition No. 34, consisting of 0.05,M cadmium sulfate hydrate, 0.1,M HEPES buffer pH 7.5 and 1.0,M sodium acetate trihydrate, with 40%(v/v) 1,4-butanediol as an additive. The crystal belonged to space group P31, with unit-cell parameters a = b = 95.62, c = 149.13,Å. Assuming the presence of a hexamer in the asymmetric unit resulted in a Matthews coefficient (VM) of 2.3,Å3,Da,1, corresponding to a solvent content of about 46%. A detailed study of this protein will yield insights into structural stability at high temperatures and should be highly relevant to the development of antibiotics and anticancer drugs targeting the biosynthesis of purine nucleotides. [source] The serine palmitoyltransferase from Sphingomonas wittichii RW1: An interesting link to an unusual acyl carrier proteinBIOPOLYMERS, Issue 9 2010Marine C. C. Raman Abstract Serine palmitoyltransferase (SPT) catalyses the first step in the de novo biosynthesis of sphingolipids (SLs). It uses a decarboxylative Claisen-like condensation reaction to couple L -serine with palmitoyl-CoA to generate a long-chain base product, 3-ketodihydrosphingosine. SLs are produced by mammals, plants, yeast, and some bacteria, and we have exploited the complete genome sequence of Sphingomonas wittichii to begin a complete analysis of bacterial sphingolipid biosynthesis. Here, we describe the enzymatic characterization of the SPT from this organism and present its high-resolution x-ray structure. Moreover, we identified an open reading frame with high sequence homology to acyl carrier proteins (ACPs) that are common to fatty acid biosynthetic pathways. This small protein was co-expressed with the SPT and we isolated and characterised the apo- and holo-forms of the ACP. Our studies suggest a link between fatty acid and sphingolipid metabolism. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 811,822, 2010. [source] Expression, purification and crystallization of Trypanosoma cruzi dihydroorotate dehydrogenase complexed with orotateACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 10 2005Daniel Ken Inaoka Dihydroorotate dehydrogenase (DHOD) catalyzes the oxidation of dihydroorotate to orotate, the fourth step and the only redox reaction in the de novo biosynthesis of pyrimidine. DHOD from Trypanosoma cruzi (TcDHOD) has been expressed as a recombinant protein in Escherichia coli and purified to homogeneity. Crystals of the TcDHOD,orotate complex were grown at 277,K by the sitting-drop vapour-diffusion technique using polyethylene glycol 3350 as a precipitant. The crystals diffract to better than 1.8,Å resolution using synchrotron radiation (, = 0.900,Å). X-ray diffraction data were collected at 100,K and processed to 1.9,Å resolution with 98.2% completeness and an overall Rmerge of 7.8%. The TcDHOD crystals belong to the orthorhombic space group P212121, with unit-cell parameters a = 67.87, b = 71.89, c = 123.27,Å. The presence of two molecules in the asymmetric unit (2 × 34,kDa) gives a crystal volume per protein weight (VM) of 2.2,Å3,Da,1 and a solvent content of 44%. [source] | ||||||||||||||||||||||