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Active Centre (active + centre)
Selected AbstractsCombined Application of Galactose Oxidase and ,- N -Acetylhexosaminidase in the Synthesis of Complex Immunoactive N -Acetyl- D -galactosaminidesADVANCED SYNTHESIS & CATALYSIS (PREVIOUSLY: JOURNAL FUER PRAKTISCHE CHEMIE), Issue 7-8 2005Pavla Fialová Abstract A high-yield preparatory procedure for the synthesis of p -nitrophenyl 2-acetamido-2-deoxy-,- D - galacto -hexodialdo-1,5-pyranoside (2) using the galactose oxidase from Dactylium dendroides in a batch reactor was developed. Enzymatic recognition of this aldehyde and the respective uronic acid 3 obtained by NaClO2 oxidation was studied using a set of 36 fungal ,- N -acetylhexosaminidases from Acremonium, Aspergillus, Penicillium and Talaromyces genera. The aldehyde 2 was readily hydrolysed by all tested ,- N -acetylhexosaminidases but neither the uronic acid 3 nor its methyl ester 4 were accepted. Molecular modelling with docking into the active centre of the ,- N -acetylhexosaminidase from Aspergillus oryzae revealed that the aldehyde 2 is processed as a C-6 geminal diol by the enzyme. The aldehyde 2 was tested for transglycosylation reactions using GlcNAc as an acceptor. The ,- N -acetylhexosaminidase from Talaromyces flavus gave the best yields (37%) of the transglycosylation product 2-acetamido-2-deoxy-,- D - galacto -hexodialdo-1,5-pyranosyl-(1,4)-2-acetamido- 2-deoxy- D -glucopyranose, which was oxidised in situ to yield the final product 2-acetamido-2-deoxy-,- D -galactopyranosyluronic acid-(1,4)-2-acetamido-2-deoxy- D -glucopyranose (6). Compounds 3 and 6 were shown to be high-affinity ligands for two natural killer cell activation receptors, NKR-P1A and CD69. For the latter receptor they turned out to be among the best ligands described so far. This increase was obviously due to the presence of a carboxy moiety. [source] Influence of Substrate Structure on PGA-Catalyzed Acylations.ADVANCED SYNTHESIS & CATALYSIS (PREVIOUSLY: JOURNAL FUER PRAKTISCHE CHEMIE), Issue 1 2005Evaluation of Different Approaches for the Enzymatic Synthesis of Cefonicid Abstract The influence of the substrate structure on the catalytic properties of penicillin G acylase (PGA) from Escherichia coli in kinetically controlled acylations has been studied. In particular, the affinity of different ,-lactam nuclei towards the active site has been evaluated considering the ratio between the rate of synthesis (vs) and the rate of hydrolysis of the acylating ester (vh1). 7-Aminocephalosporanic acid (7-ACA) and 7-amino-3-(1-sulfomethyl-1,2,3,4-tetrazol-5-yl)thiomethyl-3-cephem-4-carboxylic acid (7-SACA) showed a good affinity for the active centre of PGA. The enzymatic acylation of these nuclei with R -methyl mandelate has been studied in order to evaluate different approaches for the enzymatic synthesis of cefonicid. The best results have been obtained in the acylation of 7-SACA. Cefonicid (8) was recovered from the reaction mixture as the disodium salt in 65% yield and about 95% of purity. Furthermore, through acylation of 7-ACA, a "one-pot" chemo-enzymatic synthesis was carried out starting from cephalosporin C using three enzymes in sequence: D -amino acid oxidase (DAO), glutaryl acylase (GA) and PGA. Cefonicid disodium salt was obtained in three steps, avoiding any intermediate purification, in 35% overall yield and about 94% purity. This approach presents several advantages compared with the classical chemical processes. [source] The CHAP domain of Cse functions as an endopeptidase that acts at mature septa to promote Streptococcus thermophilus cell separationMOLECULAR MICROBIOLOGY, Issue 5 2009Séverine Layec Summary Cell separation is dependent on cell wall hydrolases that cleave the peptidoglycan shared between daughter cells. In Streptococcus thermophilus, this step is performed by the Cse protein whose depletion resulted in the formation of extremely long chains of cells. Cse, a natural chimeric enzyme created by domain shuffling, carries at least two important domains for its activity: the LysM expected to be responsible for the cell wall-binding and the CHAP domain predicted to contain the active centre. Accordingly, the localization of Cse on S. thermophilus cell surface has been undertaken by immunogold electron and immunofluorescence microscopies using of antibodies raised against the N-terminal end of this protein. Immunolocalization shows the presence of the Cse protein at mature septa. Moreover, the CHAP domain of Cse exhibits a cell wall lytic activity in zymograms performed with cell walls of Micrococcus lysodeikticus, Bacillus subtilis and S. thermophilus. Additionally, RP-HPLC analysis of muropeptides released from B. subtilis and S. thermophilus cell wall after digestion with the CHAP domain shows that Cse is an endopeptidase. Altogether, these results suggest that Cse is a cell wall hydrolase involved in daughter cell separation of S. thermophilus. [source] Comparison of the sequences of the Aspergillus nidulans hxB and Drosophila melanogaster ma-l genes with nifS from Azotobacter vinelandii suggests a mechanism for the insertion of the terminal sulphur atom in the molybdopterin cofactorMOLECULAR MICROBIOLOGY, Issue 1 2000Laïla Amrani The molybdopterin cofactor (MoCF) is required for the activity of a variety of oxidoreductases. The xanthine oxidase class of molybdoenzymes requires the MoCF to have a terminal, cyanolysable sulphur ligand. In the sulphite oxidase/nitrate reductase class, an oxygen is present in the same position. Mutations in both the ma-l gene of Drosophila melanogaster and the hxB gene of Aspergillus nidulans result in loss of activities of all molybdoenzymes that necessitate a cyanolysable sulphur in the active centre. The ma-l and hxB genes encode highly similar proteins containing domains common to pyridoxal phosphate-dependent cysteine transulphurases, including the cofactor binding site and a conserved cysteine, which is the putative sulphur donor. Key similarities were found with NifS, the enzyme involved in the generation of the iron,sulphur centres in nitrogenase. These similarities suggest an analogous mechanism for the generation of the terminal molybdenum-bound sulphur ligand. We have identified putative homologues of these genes in a variety of organisms, including humans. The human homologue is located in chromosome 18.q12. [source] The relationship between changes in the cell wall, lipid peroxidation, proliferation, senescence and cell deathPHYSIOLOGIA PLANTARUM, Issue 1 2003Gerhard Spiteller Plants and mammals contain polyunsaturated fatty acids (PUFAs) in their membranes. PUFAs belong to the most oxygen sensitive molecules encountered in nature. It would seem that nature has selected this property of PUFAs for signalling purposes: PUFAs are stored in the surface of cells and organelles not in free form but conjugated to phospho- and galactolipids. Any change in membrane structure apparently activates membrane-bound phospholipases, which cleave the conjugates. The obtained free PUFAs are substrates for lipoxygenases (LOX). These transform PUFAs to lipidhydroperoxides (LOOHs). LOOHs are converted to a great variety of secondary products. These lipid-peroxidation (LPO) products and the resulting generated products thereof represent biological signals, which do not require a preceding activation of genes. They are produced as a non-specific response to a large variety of external or internal impacts, which therefore do not need interaction with specific receptors. When, due to an external impact, e.g. attack of a microorganism, or to a change in temperature, the amount of liberated free PUFAs exceeds a certain threshold, LOX commit suicide. Thus iron ions, located in the active centre of LOX, are liberated. Iron ions react with LOOHs in the close surroundings by generating alkoxy radicals (LO.). These induce a non-enzymatic LPO. A fraction of the LO. radicals generated from linoleic acid (LPO products derived from linoleic acid play a dominant role in signalling which was previously overlooked) is converted to 2,4-dienals which induce the programmed cell death (PCD) and the hypersensitive reaction (HR). While peroxyl radicals (LOO.) generated as intermediates in the course of an enzymatic LPO are transformed within the enzyme complex to corresponding anions (LOO,), and thus lose their reactivity, peroxyl radicals generated in non-enzymatic reactions are not deactivated. They not only react by abstraction of hydrogen atoms from activated X-H bonds of molecules in their close vicinity, but also by epoxidation of double bonds and oxidation of a variety of biological molecules, causing a dramatic change in molecular structure which finally leads to cell death. As long as reducing agents, like glutathione, or compounds with free phenolic groups are available, the amount of LOOHs is kept low. Cell death is induced in a defined way by apoptosis. But when the reducing agents have been consumed, PCD seems to switch to necrotic processes. Thus proliferation is induced by minor changes at the cell membrane, while slow changes at cell membranes are linked with apoptosis (e.g. response to attack of microorganisms or drought) and necrosis (severe wounding), depending only on the amount, but not on the type, of applied stimulus. [source] Structure and substrate docking of a hydroxy(phenyl)pyruvate reductase from the higher plant Coleus blumei Benth.ACTA CRYSTALLOGRAPHICA SECTION D, Issue 5 2010Verena Janiak Hydroxy(phenyl)pyruvate reductase [H(P)PR] belongs to the family of d -isomer-specific 2-hydroxyacid dehydrogenases and catalyzes the reduction of hydroxyphenylpyruvates as well as hydroxypyruvate and pyruvate to the corresponding lactates. Other non-aromatic substrates are also accepted. NADPH is the preferred cosubstrate. The crystal structure of the enzyme from Coleus blumei (Lamiaceae) has been determined at 1.47,Å resolution. In addition to the apoenzyme, the structure of a complex with NADP+ was determined at a resolution of 2.2,Å. H(P)PR is a dimer with a molecular mass of 34,113,Da per subunit. The structure is similar to those of other members of the enzyme family and consists of two domains separated by a deep catalytic cleft. To gain insights into substrate binding, several compounds were docked into the cosubstrate complex structure using the program AutoDock. The results show two possible binding modes with similar docking energy. However, only binding mode A provides the necessary environment in the active centre for hydride and proton transfer during reduction, leading to the formation of the (R)-enantiomer of lactate and/or hydroxyphenyllactate. [source] Structures of two superoxide dismutases from Bacillus anthracis reveal a novel active centreACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 7 2005Ian W. Boucher The BA4499 and BA5696 genes of Bacillus anthracis encode proteins homologous to manganese superoxide dismutase, suggesting that this organism has an expanded repertoire of antioxidant proteins. Differences in metal specificity and quaternary structure between the dismutases of prokaryotes and higher eukaryotes may be exploited in the development of therapeutic antibacterial compounds. Here, the crystal structure of two Mn superoxide dismutases from B. anthracis solved to high resolution are reported. Comparison of their structures reveals that a highly conserved residue near the active centre is substituted in one of the proteins and that this is a characteristic feature of superoxide dismutases from the B. cereus/B. anthracis/B. thuringiensis group of organisms. [source] The structure of l -rhamnulose-1-phosphate aldolase (class II) solved by low-resolution SIR phasing and 20-fold NCS averagingACTA CRYSTALLOGRAPHICA SECTION D, Issue 5 2002Markus Kroemer The enzyme l -rhamnulose-1-phosphate aldolase catalyzes the reversible cleavage of l -rhamnulose-1-phosphate to dihydroxyacetone phosphate and l -lactaldehyde. It is a homotetramer with an Mr of 30,000 per subunit and crystallized in space group P3221. The enzyme shows a low sequence identity of 18% with the structurally known l -fuculose-1-phosphate aldolase that splits a stereoisomer in a similar reaction. Structure analysis was initiated with a single heavy-atom derivative measured to 6,Å resolution. The resulting poor electron density, a self-rotation function and the working hypothesis that both enzymes are C4 symmetric with envelopes that resemble one another allowed the location of the 20 protomers of the asymmetric unit. The crystal-packing unit was a D4 -symmetric propeller consisting of five D4 -symmetric octamers around an internal crystallographic twofold axis. Presumably, the propellers associate laterally in layers, which in turn pile up along the 32 axis to form the crystal. The non-crystallographic symmetry was used to extend the phases to the 2.7,Å resolution limit and to establish a refined atomic model of the enzyme. The structure showed that the two enzymes are indeed homologous and that they possess chemically similar active centres. [source] | |||||||||||||