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Substrate D (substrate + d)
Selected Abstracts5-hydroxytryptamine-mediated increase in glutamate uptake by the leech giant glial cellGLIA, Issue 8 2006Ingolf C. Hirth Abstract The clearance of synaptically released glutamate is one of the pivotal functions of glial cells. We have studied the role of 5-hydroxytryptamine (5-HT, 30 ,M), a neurotransmitter and neurohormone in the leech central nervous system with a versatile action spectrum, on the efficacy of glial glutamate uptake. The activity of the glutamate uptake carrier in the giant glial cell in isolated ganglia of Hirudo medicinalis was monitored by measuring the membrane current and the change in the intracellular Na+ concentration (Na+i) as induced by the glutamate carrier substrate D -aspartate (D -asp, 1 mM). 5-HT increased the D -asp-induced current (EC50 at 5 ,M) and rise in Na+i, an effect which was mimicked by the membrane-permeable cyclic nucleotide analogue dibutyryl-cyclic AMP (db-cAMP). The adenylyl cyclase inhibitor SQ 22,536 and the protein kinase A antagonist Rp-cAMP inhibited the effect of 5-HT. Blocking the G protein in the giant glial cell by injecting GDP-,-S suppressed the effect of 5-HT, but not the effect of db-cAMP, on the D -asp-induced current. Our results suggest that 5-HT enhances the glial uptake of glutamate via cAMP- and PKA-mediated pathway. © 2006 Wiley-Liss, Inc. [source] Preliminary X-ray crystallographic analysis of the d -xylulose 5-phosphate phosphoketolase from Lactococcus lactisACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 7 2010Georgiana Petrareanu Phosphoketolases are thiamine diphosphate-dependent enzymes which play a central role in the pentose-phosphate pathway of heterofermentative lactic acid bacteria. They belong to the family of aldehyde-lyases and in the presence of phosphate ion cleave the carbon,carbon bond of the specific substrate d -xylulose 5-phosphate (or d -fructose 6-phosphate) to give acetyl phosphate and d -glyceraldehyde 3-phosphate (or d -erythrose 4-phosphate). Structural information about phosphoketolases is particularly important in order to fully understand their mechanism as well as the steric course of phosphoketolase-catalyzed reactions. Here, the purification, preliminary crystallization and crystallographic characterization of d -xylulose 5-phosphate phosphoketolase from Lactococcus lactis are reported. The presence of thiamine diphosphate during purification was essential for the enzymatic activity of the purified protein. The crystals belonged to the monoclinic space group P21. Diffraction data were obtained to a resolution of 2.2,Å. [source] Structure of d -3-hydroxybutyrate dehydrogenase prepared in the presence of the substrate d -3-hydroxybutyrate and NAD+ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 4 2009Md Mominul Hoque d -3-Hydroxybutyrate dehydrogenase from Alcaligenes faecalis catalyzes the reversible conversion between d -3-hydroxybutyrate and acetoacetate. The enzyme was crystallized in the presence of the substrate d -3-hydroxybutyrate and the cofactor NAD+ at the optimum pH for the catalytic reaction. The structure, which was solved by X-ray crystallography, is isomorphous to that of the complex with the substrate analogue acetate. The product as well as the substrate molecule are accommodated well in the catalytic site. Their binding geometries suggest that the reversible reactions occur by shuttle movements of a hydrogen negative ion from the C3 atom of the substrate to the C4 atom of NAD+ and from the C4 atom of NADH to the C3 atom of the product. The reaction might be further coupled to the withdrawal of a proton from the hydroxyl group of the substrate by the ionized Tyr155 residue. These structural features strongly support the previously proposed reaction mechanism of d -3-hydroxybutyrate dehydrogenase, which was based on the acetate-bound complex structure. [source] Model-based characterization of an amino acid racemase from Pseudomonas putida DSM 3263 for application in medium-constrained continuous processesBIOTECHNOLOGY & BIOENGINEERING, Issue 4 2007M. Bechtold Abstract The amino acid racemase with broad substrate specificity from Pseudomonas putida DSM 3263 was overproduced and characterized with respect to application in an integrated multi-step process (e.g., dynamic kinetic resolution) that,theoretically,would allow for 100% chemical yield and 100% enantiomeric excess. Overexpression of the racemase gene in Escherichia coli delivered cell free extract with easily sufficient activity (20,50 U,mg,1 total protein) for application in an enzyme membrane reactor (EMR) setting. Model-based experimental analysis of a set of enzyme assays clearly indicated that racemization of the model substrates D - or L -methionine could be accurately described by reversible Michaelis,Menten kinetics. The corresponding kinetic parameters were determined from progress curves for the entire suitable set of aqueous-organic mixtures (up to 60% methanol and 40% acetonitrile) that are eligible for an integrated process scheme. The resulting kinetic expression could be successfully applied to describe enzyme membrane reactor performance under a large variety of settings. Model-based calculations suggested that a methanol content of 10% and an acetonitrile content of 20% provide maximum productivity in EMR operations. However product concentrations were decreased in comparison to purely aqueous operation due to decreasing solubility of methionine with increasing organic solvent content. Finally, biocatalyst stability was investigated in different solvent compositions following a model-based approach. Buffer without organic content provided excellent stability at moderate temperatures (20,35°C) while addition of 20% acetonitrile or methanol drastically reduced the half-life of the racemase. Biotechnol. Bioeng. 2007; 98: 812,824. © 2007 Wiley Periodicals, Inc. [source] | ||||||||||