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Extradiol oxidative cleavage of catechols by ferrous and ferric complexes of 1,4,7-triazacyclononane: Insight into the mechanism of the extradiol catechol dioxygenases
No full textThe major oxygenation product of catechol by dioxygen in the presence of FeCl2 or FeCl3, 1,4,7-triazacyclononane (TACN), and pyridine in methanol is the extradiol cleavage product 2-hydroxymuconic semi-aldehyde methyl ester (Lin, G.; Reid, G.; Bugg, T. D. I-I. J. Chem. Sec. Chem. Commun. 2000, 1119-1120). Under these conditions, extradiol cleavage of a range of 3- and 4-substituted catechols with electron-donating substituents is observed. The reaction shows a preference in selectivity and rate for iron(II) rather than iron(III) for the extradiol cleavage, which parallels the selectivity of the extradiol dioxygenase family. The reaction also shows a high selectivity for the macrocyclic ligand, TACN, over a range of other nitrogen-and oxygen-containing macrocycles. Reaction of anaerobically prepared iron-TACN complexes with dioxygen gave the same product as monitored by UV/vis spectroscopy. KO2 is able to oxidize catechols with both electron-donating and electron-withdrawing substituents, implying a different mechanism for extradiol. cleavage. Saturation kinetics were observed for catechols, which fit the Michaelis-Menten equation to give k(cat)(app) = 4.8 x 10(-3) s(-1) for 3-(2' ,3'-dihydroxyphenyl)propionic acid. The reaction was also found to proceed using monosodium catecholate in the absence of pyridine, but with different product ratios, giving insight into the acid/base chemistry of extradiol cleavage. In particular, extradiol cleavage in the presence of iron(II) shows a requirement for a proton donor, implying a role for an acidic group in the extradiol dioxygenase active site
Cis-trans isomerization of a cyclopropyl radical trap catalyzed by extradiol catechol dioxygenases: evidence for a semiquinone intermediate
No full textSubstrate analogues cis- and trans-2-(2,3-dihydroxyphenyl)cyclopropane-1-carboxylic acid were synthesized as probes for a semiquinone radical intermediate in the (2,3-dihydroxyphenyl)propionate 1,2-dioxygenase reaction. These analogues were found to be substrates for oxidative cleavage by extradiol dioxygenases from Escherichia coli and Alcaligenes eutrophus. The stereochemistry of the ring fission products was analyzed by conversion to cyclopropane-1,2-dicarboxylic acids using the ensuing hydrolase enzyme MhpC, followed by GCMS analysis. This analysis revealed 85-94% trans product and 6-15% cis products, implying that cis/trans isomerization of the cyclopropyl ring substituents had taken place during the enzymatic conversion. These results are consistent with a reversible opening of the cyclopropyl ring, and hence consistent with the intermediacy of a semiquinone radical intermediate in the extradiol catechol dioxygenase reaction.</p
Mechanism of extradiol catechol dioxygenases: evidence for a lactone intermediate in the 2,3-dihydroxyphenylpropionate 1,2-dioxygenase reaction
No full textIn lieu of an abstract, this is the article's first paragraph. The oxidative cleavage of catechols by non-heme iron-dependent dioxygenase enzymes is a key step in the bacterial degradation of naturally-occurring and man-made aromatic compounds.1 Two classes of catechol dioxygenases are found: iron(UI)-dependent intradiol dioxygenases, which cleave the carbon—carbon bond between the two hydroxyl groups, and iron(II)-dependent extradiol dioxygenases, which cleave a carbon—carbon bond adjacent to the two hydroxyl groups. Despite extensive spectroscopic studies on these enzymes and the determination of the crystal structure of protocatechuate 3,4-dioxygenase,3 45only limited data are available regarding the mechanism of carbon—carbon bond cleavage. A dioxetane intermediate was originally proposed for the .intradiol enzyme catechol 1,2-dioxygenase based on 1S02 labeling studies; however, more recently an anhydride intermediate has been proposed for the intradiol class, formed by a Criegee rearrangement. In view of the key environmental significance of the catechol dioxygenases and the absence of mechanistic information regarding the extradiol enzymes, we have initiated a study of the mechanism of iron(II)-dependent 2,3-dihydroxyphenyl-propionate 1,2-dioxygenase (MhpB) from Escherichia coli. Here we report evidence from 18G labeling studies and analogue synthesis for a lactone intermediate
Enzymatic breakdown of poly-?-D-glutamic acid in Bacillus licheniformis: Identification of a polyglutamyl ?-hydrolase enzyme
No full textA polyglutamyl gamma -hydrolase enzyme has been identified which catalyses the hydrolytic breakdown of poly-gamma -D-glutamic acid (PGA) from Bacillus licheniformis 9945a. The enzyme was found to be physically associated with the polymer and was activated by Zn2+ or Ca2+ salts. The enzyme can be solubilized from the polymer by treatment with 0.5% SDS and 1 mM ZnCl2 and can then be renatured onto exogenous PGA upon dilution below the detergent critical micellar concentration. The enzyme was partially purified by affinity chromatography, using immobilized PGA. Peptide thioesters containing one and two gamma -glutamyl units were synthesized as potential chromogenic substrates but showed no activity with the solubilized enzyme. Examination of C-14-labeled reaction products indicated that the enzyme is an endo-type hydrolase
2-hydroxy-6-keto-nona-2,4-diene 1,9-dioic acid 5,6-hydrolase: evidence from O-18 isotope exchange for gem-diol intermediate
No full textThe mechanism-based inactivation and subsequent identification of the nucleophilic residue using mass spectrometry have been successfully applied and used to identify the active-site nucleophile in numerous ?-glycosidases, as illustrated using C. fimi exoglycanase. Evidence for a covalent glycosyl-enzyme intermediate has come from X-ray crystallographic analysis of trapped complexes, the first being that of the trapped fluoroglycosyl-enzyme intermediate of Cex. The crystal structure of the trapped fluorocellobiosyl-enzyme complex for Cex has provided useful insights into catalysis and the roles of specific residues at the active site. In addition, information about the conformation of the natural sugar in the covalently bound state and the interactions at the active site was obtained using a mutant form of Cex
Multivalent conjugates of poly-?-D-glutamic acid from Bacillus licheniformis with antibody F(ab') and glycopeptide ligands
No full textPoly--D-glutamic acid from Bacillus licheniformis is a water-soluble, nontoxic, nonimmunogenic exopolymer. Using synthetic linkers, the -carboxylate side chains of PGA were conjugated to an exposed thiol side chain of an antibody F(ab') fragment, Mc109F4. Analysis of the PGA-Mc109F4 conjugate by gel filtration HPLC revealed a mixture of multivalent conjugates. The PGA-Mc109F4 conjugate retained biological activity, but showed a lower binding affinity to target BCL3B3 cells than free Mc109F4 F(ab')2 by flow cytometry, and a lower efficacy for BCL3B3 growth inhibition than free Mc109F4 F(ab')2. PGA was also conjugated with the free amino group of glycopeptide antibiotic vancomycin. The PGA-vancomycin conjugate showed slightly lower antibacterial activity than free vancomycin versus susceptible Bacillus subtilis, but slightly higher activity versus intrinsically resistant Leuconostoc mesenteroides
Biological properties of N-acyl and N-haloacetyl neuraminic acids: processing by enzymes of sialic acid metabolism, and interaction with influenza virus
No full textSeveral unnatural N-acyl neuraminic acids (N-propionyl, N-hexanoyl, N-benzoyl, N-trifluoroacetyl, N-chloroacetyl, N-difluoroacetyl) were prepared enzymatically using immobilised sialic acid aldolase. N-Trifluoroacetyl- N-chloroacetyl- and N-difluoroacetyl neuraminic acids were shown to enhance up to 10-fold the rate of association of influenza virus A to a sialoglycolipid neomembrane by surface plasmon resonance, and were found to act as weak inhibitors (K-iapp 0.45-2.0 mM) of influenza virus neuraminidase. The N-propionyl, N-chloroacetyl- and N-difluoroacetyl neuraminic acids were found to be substrates for recombinant Escherichia coli CMP sialate synthase, to give the corresponding CMP-N-acyl-neuraminic acids. CMP-N-propionyl neuraminic acid was found not to be a substrate for CMP-N-acetyl neuraminic acid hydroxylase from pig submandibular gland
Phospho-N-acetyl-muramyl-pentapeptide translocase from Escherichia coli: catalytic role of conserved aspartic acid residues
No full textPhospho-N-acetyl-muramyl-pentapeptide translocase (translocase 1) catalyzes the first of a sequence of lipid-linked steps that ultimately assemble the peptidoglycan layer of the bacterial cell wall. This essential enzyme is the target of several natural product antibiotics and has recently been the focus of antimicrobial drug discovery programs. The catalytic mechanism of translocase 1 is believed to proceed via a covalent intermediate formed between phospho-N-acetyl-muramyl-pentapeptide and a nucleophilic amino acid residue. Amino acid sequence alignments of the translocase 1 family and members of the related transmembrane phosphosugar transferase superfamily revealed only three conserved residues that possess nucleophilic side chains: the aspartic acid residues D115, D116, and D267. Here we report the expression and partial purification of Escherichia coli translocase 1 as a C-terminal hexahistidine (C-His(6)) fusion protein. Three enzymes with the site-directed mutations D115N, D116N, and D267N were constructed, expressed, and purified as C-His, fusions. Enzymatic analysis established that all three mutations eliminated translocase I activity, and this finding verified the essential role of these residues. By analogy with the structural environment of the double aspartate motif found in prenyl transferases, we propose a model whereby D115 and D116 chelate a magnesium ion that coordinates with the pyrophosphate bridge of the UDP-N-acetyl-muramyl-pentapeptide substrate and in which D267 therefore fulfills the role of the translocase 1 active-site nucleophile
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
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