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Thiamine Diphosphate (thiamine + diphosphate)
Selected AbstractsThe crystal structure of phenylpyruvate decarboxylase from Azospirillum brasilense at 1.5 Å resolutionFEBS JOURNAL, Issue 9 2007Implications for its catalytic, regulatory mechanism Phenylpyruvate decarboxylase (PPDC) of Azospirillum brasilense, involved in the biosynthesis of the plant hormone indole-3-acetic acid and the antimicrobial compound phenylacetic acid, is a thiamine diphosphate-dependent enzyme that catalyses the nonoxidative decarboxylation of indole- and phenylpyruvate. Analogous to yeast pyruvate decarboxylases, PPDC is subject to allosteric substrate activation, showing sigmoidal v versus [S] plots. The present paper reports the crystal structure of this enzyme determined at 1.5 Å resolution. The subunit architecture of PPDC is characteristic for other members of the pyruvate oxidase family, with each subunit consisting of three domains with an open ,/, topology. An active site loop, bearing the catalytic residues His112 and His113, could not be modelled due to flexibility. The biological tetramer is best described as an asymmetric dimer of dimers. A cysteine residue that has been suggested as the site for regulatory substrate binding in yeast pyruvate decarboxylase is not conserved, requiring a different mechanism for allosteric substrate activation in PPDC. Only minor changes occur in the interactions with the cofactors, thiamine diphosphate and Mg2+, compared to pyruvate decarboxylase. A greater diversity is observed in the substrate binding pocket accounting for the difference in substrate specificity. Moreover, a catalytically important glutamate residue conserved in nearly all decarboxylases is replaced by a leucine in PPDC. The consequences of these differences in terms of the catalytic and regulatory mechanism of PPDC are discussed. [source] Active-site changes in the pyruvate dehydrogenase multienzyme complex E1 apoenzyme component from Escherichia coli observed at 2.32,Å resolutionACTA CRYSTALLOGRAPHICA SECTION D, Issue 11 2006Palaniappa Arjunan The first enzymatic component, E1 (EC 1.2.4.1), of the pyruvate dehydrogenase multienzyme complex (PDHc) utilizes thiamine diphosphate (ThDP) and Mg2+ as cofactors. The structure of a branched-chain-specific E1 apoenzyme from the heterotetrameric ,2,2 E1 family was recently reported and showed that disorder-to-order transformations in two active-site loops take place upon cofactor binding. To ascertain what effect the absence of cofactor may have in the homodimeric ,2Escherichia coli PDHc E1, the corresponding apoenzyme has been prepared and its three-dimensional structure determined and analyzed at 2.32,Å by crystallographic methods. This represents the first reported apoenzyme structure for any E1 component from the homodimeric ,2 family. Electron-density features occurring in the region where the cofactor pyrimidine ring would normally be expected to bind are of size, shape and location compatible with water molecules that form a hydrogen-bonded linkage between residues Glu571 and Val192, which normally make conserved interactions with the ThDP cofactor. A histidine side chain that normally forms hydrogen bonds to ThDP is disordered in its absence and partially occupies two sites. Unlike in the reported heterotetrameric branched-chain apo-E1, no disorder/order loop transformations are evident in apo-PDHc E1 relative to the holo-E1 enzyme (PDHc E1,ThDP,Mg2+). Differences in the extent of hydrogen-bonding networks found in the apo-E1 enzyme, the holo-E1 enzyme and in an inhibitor complex with bound thiamine 2-thiazolone diphosphate (ThTDP), PDHc E1,ThTDP,Mg2+, are described. [source] Overexpression, crystallization and preliminary X-ray analysis of xylulose-5-phosphate/fructose-6-phosphate phosphoketolase from Bifidobacterium breveACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 8 2010Ryuichiro Suzuki The xylulose-5-phosphate/fructose-6-phosphate phosphoketolase gene from Bifidobacterium breve was cloned and overexpressed in Escherichia coli. The enzyme was purified to homogeneity and crystallized by the sitting-drop vapour-diffusion method. Crystals were obtained at 293,K using 0.05,mM thiamine diphosphate, 0.25,mM MgCl2, 24%(w/v) PEG 6000 and 0.1,M Bicine pH 9.0. The crystals belonged to the tetragonal space group I422, with unit-cell parameters a = b = 174.8, c = 163.8,Å, and diffracted to beyond 1.7,Å resolution. [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] The structures of pyruvate oxidase from Aerococcus viridans with cofactors and with a reaction intermediate reveal the flexibility of the active-site tunnel for catalysisACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 11 2007Ella Czarina Magat Juan The crystal structures of pyruvate oxidase from Aerococcus viridans (AvPOX) complexed with flavin adenine dinucleotide (FAD), with FAD and thiamine diphosphate (ThDP) and with FAD and the 2-acetyl-ThDP intermediate (AcThDP) have been determined at 1.6, 1.8 and 1.9,Å resolution, respectively. Each subunit of the homotetrameric AvPOX enzyme consists of three domains, as observed in other ThDP-dependent enzymes. FAD is bound within one subunit in the elongated conformation and with the flavin moiety being planar in the oxidized form, while ThDP is bound in a conserved V-conformation at the subunit,subunit interface. The structures reveal flexible regions in the active-site tunnel which may undergo conformational changes to allow the entrance of the substrates and the exit of the reaction products. Of particular interest is the role of Lys478, the side chain of which may be bent or extended depending on the stage of catalysis. The structures also provide insight into the routes for electron transfer to FAD and the involvement of active-site residues in the catalysis of pyruvate to its products. [source] Structure of the ThDP-dependent enzyme benzaldehyde lyase refined to 1.65,Å resolutionACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 7 2007Andy Maraite Benzaldehyde lyase (BAL; EC 4.1.2.38) is a thiamine diphosphate (ThDP) dependent enzyme that catalyses the enantioselective carboligation of two molecules of benzaldehyde to form (R)-benzoin. BAL has hence aroused interest for its potential in the industrial synthesis of optically active benzoins and derivatives. The structure of BAL was previously solved to a resolution of 2.6,Å using MAD experiments on a selenomethionine derivative [Mosbacher et al. (2005), FEBS J.272, 6067,6076]. In this communication of parallel studies, BAL was crystallized in an alternative space group (P212121) and its structure refined to a resolution of 1.65,Å, allowing detailed observation of the water structure, active-site interactions with ThDP and also the electron density for the co-solvent 2-methyl-2,4-pentanediol (MPD) at hydrophobic patches of the enzyme surface. [source] | ||||||||||