scholarly journals Novel intersubunit active site of arcchaeal N,N′-diacetylchitobiose deacetylase

2014 ◽  
Vol 70 (a1) ◽  
pp. C473-C473
Author(s):  
Tsutomu Nakamura ◽  
Mayumi Niiyama ◽  
Wakana Hashimoto ◽  
Takahisa Ikegami ◽  
Daisuke Koma ◽  
...  
Keyword(s):  
Active Site ◽  
Target Genes ◽  
Side Chain ◽  
Expression Plasmid ◽  
E Coli ◽  
Reservoir Solution ◽  
Acetyl Group ◽  
Hydrolysis Of ◽  
Catalytic Zinc ◽  
Soluble Recombinant Protein

N,N′-diacetylchitobiose deacetylase (Dac) is involved in the archaea-specific chitinolytic pathway. In order to develop a structure-based understanding of the chitinolytic pathway in hyperthermophilic Pyrococcus species, we performed crystallographic studies on Dacs from P. horikoshii (Ph-Dac) and P. furiosus (Pf-Dac). Neither Ph-Dac nor Pf-Dac was expressed in the soluble fraction of Escherichia coli harboring the expression plasmid. However, insertion of the target genes into the chromosome of E. coli yielded the soluble recombinant protein. The purified Pyrococcus Dacs were thermostable up to 950C. The crystal structures of Ph-Dac and Pf-Dac were determined at resolutions of 2.0 Å and 1.54 Å, respectively. The Pyrococcus Dac forms a hexamer comprised of two trimers. These Dacs are characterized by an intermolecular cleft, which is formed by two polypeptides in the trimeric assembly. In Ph-Dac, catalytic zinc situated at the end of the cleft is coordinated by three side chain ligands from His44, Asp47, and His155, and by a phosphate ion derived from the crystallization reservoir solution. We considered that the bound phosphate mimicked the tetrahedral oxyanion, which is an intermediate of hydrolysis of the N-acetyl group, and proposed an appropriate reaction mechanism. In the proposed mechanism, the Nε atom of His264 (from the adjacent polypeptide in the Ph-Dac sequence) is directly involved in the stabilization of the oxyanion intermediate. These factors give the archaeal Dacs unprecedented active site architecture as a zinc-dependent deacetylase.

scholarly journals Interaction of L-threo and L-erythro isomers of 3-fluoroglutamate with glutamate decarboxylase from Escherichia coli

1985 ◽  
Vol 229 (3) ◽  
pp. 675-678 ◽  
Author(s):  
A Vidal-Cros ◽  
M Gaudry ◽  
A Marquet
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Glutamate Decarboxylase ◽  
Fluorine Atom ◽  
Pyridoxal Phosphate ◽  
Succinic Semialdehyde ◽  
E Coli ◽  
Rigid Conformation ◽  
The Difference ◽  
Hydrolysis Of

L-threo-3-Fluoroglutamate and L-erythro-3-fluoroglutamate were tested with glutamate decarboxylase from Escherichia coli. Both isomers were substrates: the threo isomer was decarboxylated into optically active 4-amino-3-fluorobutyrate, whereas the erythro isomer lost the fluorine atom during the reaction, yielding succinic semialdehyde after hydrolysis of the unstable intermediate enamine. The difference between the two isomers demonstrates that the glutamic acid-pyridoxal phosphate Schiff base is present at the active site under a rigid conformation. Furthermore, although the erythro isomer lost the fluorine atom, yielding a reactive aminoacrylic acid in the active site, no irreversible inactivation of E. coli glutamate decarboxylase was observed.

scholarly journals Pseudomonas aeruginosa Exopolyphosphatase Is Also a Polyphosphate: ADP Phosphotransferase

2015 ◽  
Vol 2015 ◽  
pp. 1-13 ◽  
Author(s):  
Paola R. Beassoni ◽  
Lucas A. Gallarato ◽  
Cristhian Boetsch ◽  
Mónica N. Garrido ◽  
Angela T. Lisa
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Enzyme Activity ◽  
Active Site ◽  
Inorganic Phosphate ◽  
Biochemical Characterization ◽  
E Coli ◽  
Activity Dependent ◽  
Hydrolysis Of ◽  
Molecular Modeling And Docking

Pseudomonas aeruginosa exopolyphosphatase (paPpx; EC 3.6.1.11) catalyzes the hydrolysis of polyphosphates (polyP), producing polyPn−1 plus inorganic phosphate (Pi). In a recent work we have shown that paPpx is involved in the pathogenesis of P. aeruginosa. The present study was aimed at performing the biochemical characterization of this enzyme. We found some properties that were already described for E. coli Ppx (ecPpx) but we also discovered new and original characteristics of paPpx: (i) the peptide that connects subdomains II and III is essential for enzyme activity; (ii) NH4+ is an activator of the enzyme and may function at concentrations lower than those of K+; (iii) Zn2+ is also an activator of paPpx and may substitute Mg2+ in the catalytic site; and (iv) paPpx also has phosphotransferase activity, dependent on Mg2+ and capable of producing ATP regardless of the presence or absence of K+ or NH4+ ions. In addition, we detected that the active site responsible for the phosphatase activity is also responsible for the phosphotransferase activity. Through the combination of molecular modeling and docking techniques, we propose a model of the paPpx N-terminal domain in complex with a polyP chain of 7 residues long and a molecule of ADP to explain the phosphotransferase activity.

A designed supramolecular protein assembly with in vivo enzymatic activity

Science ◽  
2014 ◽  
Vol 346 (6216) ◽  
pp. 1525-1528 ◽  
Author(s):  
Woon Ju Song ◽  
F. Akif Tezcan
Keyword(s):  
Active Site ◽  
First Principles ◽  
Self Assembly ◽  
Protein Structures ◽  
Three Dimensional ◽  
In Vivo Screening ◽  
Hydrolysis Of ◽  
Catalytic Zinc ◽  
Tetrameric Assembly

The generation of new enzymatic activities has mainly relied on repurposing the interiors of preexisting protein folds because of the challenge in designing functional, three-dimensional protein structures from first principles. Here we report an artificial metallo-β-lactamase, constructed via the self-assembly of a structurally and functionally unrelated, monomeric redox protein into a tetrameric assembly that possesses catalytic zinc sites in its interfaces. The designed metallo-β-lactamase is functional in the Escherichia coli periplasm and enables the bacteria to survive treatment with ampicillin. In vivo screening of libraries has yielded a variant that displays a catalytic proficiency [(kcat/Km)/kuncat] for ampicillin hydrolysis of 2.3 × 106 and features the emergence of a highly mobile loop near the active site, a key component of natural β-lactamases to enable substrate interactions.

scholarly journals Biophysical characterization of two commercially available preparations of the drug containing Escherichia coli L-Asparaginase 2

2020 ◽  
Author(s):  
Talita Stelling de Araújo ◽  
Sandra M. N. Scapin ◽  
William de Andrade ◽  
Maira Fasciotti ◽  
Mariana T. Q. de Magalhães ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Lymphoblastic Leukemia ◽  
Biophysical Characterization ◽  
E Coli ◽  
Terminal Loop ◽  
Hydrolysis Of

AbstractThe hydrolysis of asparagine and glutamine by L-asparaginase has been used to treat acute lymphoblastic leukemia for over four decades. Each L-asparaginase monomer has a long loop that closes over the active site upon substrate binding, acting as a lid. Here we present a comparative study two commercially available preparations of the drug containing Escherichia coli L-Asparaginase 2, performed by a comprehensive array of biophysical and biochemical approaches. We report the oligomeric landscape and conformational and dynamic plasticity of E. coli type 2 L-asparaginase (EcA2) present in two different formulations, and its relationship with L-aspartic acid, which is present in Aginasa, but not in Leuginase. EcA2 shows a composition of monomers and oligomers up to tetramers, which is mostly not altered in the presence of L-Asp. The N-terminal loop of Leuginase, which is part of the active site is flexibly disordered, but gets ordered as in Aginasa in the presence os L-Asp, while L-Glu only does so to a limited extent. Ion-mobility spectrometry–mass spectrometry reveals two conformers for the monomeric EcA2, one of which can selectively bind to L-Asp and L-Glu. Aginasa has higher resistance to in vitro proteolysis than Leuginase, and this is directly related to the presence of L-Asp.

scholarly journals Structural analysis of E. coli 6-carboxytetrahydropterin synthase

2014 ◽  
Vol 70 (a1) ◽  
pp. C462-C462
Author(s):  
Asaithambi Killivalavan ◽  
Kyung Seo ◽  
Ningning Zhuang ◽  
Young Park ◽  
Kon Lee
Keyword(s):  
Active Site ◽  
Essential Role ◽  
Site Directed Mutagenesis ◽  
Side Chain ◽  
Wild Type ◽  
Specific Enzyme ◽  
Aromatic Residues ◽  
E Coli

The Escherichia coli 6-carboxytetrahydropterin synthase (eCTPS), a homolog of 6-pyruvoyl tetrahydropterin synthase (PTPS), possesses a much stronger catalytic activity to cleave the side chain of sepiapterin in vitro rather than the genuine PTPS activity and catalyzes the conversion of dihydroneopterin triphosphate to 6-carboxy-5,6,7,8-tetrahydropterin in vivo. We have determined crystal structures of a wild type apo-eCTPS and a Cys27Ala mutant eCTPS complexed with sepiapterin up to 2.3 and 2.5 Å, respectively. The structures are highly conserved at the active site and the Zn2+ binding site. However, comparison of the eCTPS structures with those of mammalian PTPS homologs revealed that two specific residues Trp51 and Phe55, not existing in the mammalian PTPS, kept the substrate bound by stacking it with their side chains. Replacements of these two residues by site-directed mutagenesis to the residues, Met and Leu, existing only in mammalian PTPS, converted the eCTPS to have the mammalian PTPS activity. Our studies confirm that these two aromatic residues in eCTPS play an essential role in stabilizing the substrate and for the specific enzyme activity different from the original PTPS activity. These aromatic residues Trp51 and Phe55 are a key signature of bacterial PTPS enzymes that distinguish them from mammalian PTPS homologs.

Enhanced Biocatalytic Activity of Recombinant Lipase Immobilized on Gold Nanoparticles

2019 ◽  
Vol 20 (6) ◽  
pp. 497-505 ◽  
Author(s):  
Abeer M. Abd El-Aziz ◽  
Mohamed A. Shaker ◽  
Mona I. Shaaban
Keyword(s):  
Gold Nanoparticles ◽  
Lipolytic Activity ◽  
Market Demand ◽  
Biotechnological Applications ◽  
Lipase Gene ◽  
Expression Plasmid ◽  
E Coli ◽  
Nickel Affinity Chromatography ◽  
Recombinant Production ◽  
Sds Page

Background: Bacterial lipases especially Pseudomonas lipases are extensively used for different biotechnological applications. Objectives: With the better understanding and progressive needs for improving its activity in accordance with the growing market demand, we aimed in this study to improve the recombinant production and biocatalytic activity of lipases via surface conjugation on gold nanoparticles. Methods: The full length coding sequences of lipase gene (lipA), lipase specific foldase gene (lipf) and dual cassette (lipAf) gene were amplified from the genomic DNA of Pseudomonas aeruginosa PA14 and cloned into the bacterial expression vector pRSET-B. Recombinant lipases were expressed in E. coli BL-21 (DE3) pLysS then purified using nickel affinity chromatography and the protein identity was confirmed using SDS-PAGE and Western blot analysis. The purified recombinant lipases were immobilized through surface conjugation with gold nanoparticles and enzymatic activity was colorimetrically quantified. Results: Here, two single expression plasmid systems pRSET-B-lipA and pRSET-B-lipf and one dual cassette expression plasmid system pRSET-B-lipAf were successfully constructed. The lipolytic activities of recombinant lipases LipA, Lipf and LipAf were 4870, 426 and 6740 IUmg-1, respectively. However, upon immobilization of these recombinant lipases on prepared gold nanoparticles (GNPs), the activities were 7417, 822 and 13035 IUmg-1, for LipA-GNPs, Lipf-GNPs and LipAf-GNPs, respectively. The activities after immobilization have been increased 1.52 and 1.93 -fold for LipA and LipAf, respectively. Conclusion: The lipolytic activity of recombinant lipases in the bioconjugate was significantly increased relative to the free recombinant enzyme where immobilization had made the enzyme attain its optimum performance.

Synthesis of Normuramic Acid Carba Analog and Its Glycopeptide Derivative Resistant to β-Elimination Splitting of the Side Chain

2000 ◽  
Vol 65 (11) ◽  
pp. 1726-1736 ◽  
Author(s):  
Miroslav Ledvina ◽  
Radka Pavelová ◽  
Anna Rohlenová ◽  
Jan Ježek ◽  
David Šaman
Keyword(s):  
Ethyl Ester ◽  
Alkaline Hydrolysis ◽  
Benzyl Ester ◽  
Side Chain ◽  
Propanoic Acid ◽  
Hydrolysis Of

Carba analogs of normuramic acid, i.e., 3-(benzyl 2-acetamido-2,3-dideoxy-4,6-O-isopropylidene-α-D-glucopyranosid-3-yl)propanoic acid derivatives (nitrile or esters) 3a-3c were prepared by addition of radicals generated from benzyl 2-acetamido-2-deoxy-4,6-O-isopropylidene-3-O-[(methylsulfanyl)thiocarbonyl]- (2a) or -3-O-(phenoxythiocarbonyl)-α-D-glucopyranoside (2b) with Bu3SnH to acrylonitrile or acryl esters. Alkaline hydrolysis of ethyl ester 3c afforded 3-(benzyl 2-acetamido-2,3-dideoxy-4,6-O-isopropylidene-α-D-glucopyranosid-3-yl)propanoic acid (5). Coupling of acid 5 with L-2-aminobutanoyl-D-isoglutamine benzyl ester trifluoroacetate and subsequent deprotection of the intermediate 6 furnished N-[3-(2-acetamido-2,3-dideoxy-α-D-glucopyranosid-3-yl)propanoyl]-L-2-aminobutanoyl-D-isoglutamine (7).

scholarly journals “Newton’s cradle” proton relay with amide–imidic acid tautomerization in inverting cellulase visualized by neutron crystallography

2015 ◽  
Vol 1 (7) ◽  
pp. e1500263 ◽  
Author(s):  
Akihiko Nakamura ◽  
Takuya Ishida ◽  
Katsuhiro Kusaka ◽  
Taro Yamada ◽  
Shinya Fushinobu ◽  
...  
Keyword(s):  
Glycoside Hydrolases ◽  
Side Chain ◽  
Enzymatic Reactions ◽  
X Ray Diffraction ◽  
Peptide Bonds ◽  
Proton Relay ◽  
Newton’S Cradle ◽  
Dynamics Simulations ◽  
Hydrolysis Of

Hydrolysis of carbohydrates is a major bioreaction in nature, catalyzed by glycoside hydrolases (GHs). We used neutron diffraction and high-resolution x-ray diffraction analyses to investigate the hydrogen bond network in inverting cellulase PcCel45A, which is an endoglucanase belonging to subfamily C of GH family 45, isolated from the basidiomycete Phanerochaete chrysosporium. Examination of the enzyme and enzyme-ligand structures indicates a key role of multiple tautomerizations of asparagine residues and peptide bonds, which are finally connected to the other catalytic residue via typical side-chain hydrogen bonds, in forming the “Newton’s cradle”–like proton relay pathway of the catalytic cycle. Amide–imidic acid tautomerization of asparagine has not been taken into account in recent molecular dynamics simulations of not only cellulases but also general enzyme catalysis, and it may be necessary to reconsider our interpretation of many enzymatic reactions.

scholarly journals Binding Site of the Bridged Macrolides in the Escherichia coli Ribosome

2005 ◽  
Vol 49 (1) ◽  
pp. 281-288 ◽  
Author(s):  
Liqun Xiong ◽  
Yakov Korkhin ◽  
Alexander S. Mankin
Keyword(s):  
Escherichia Coli ◽  
Tight Binding ◽  
Dimethyl Sulfate ◽  
23S Rrna ◽  
Side Chain ◽  
Macrolide Antibiotics ◽  
Side Chains ◽  
Alkyl Aryl ◽  
E Coli ◽  
Exit Tunnel

ABSTRACT Ketolides represent the latest group of macrolide antibiotics. Tight binding of ketolides to the ribosome appears to correlate with the presence of an extended alkyl-aryl side chain. Recently developed 6,11-bridged bicyclic ketolides extend the spectrum of platforms used to generate new potent macrolides with extended alkyl-aryl side chains. The purpose of the present study was to characterize the site of binding and the action of bridged macrolides in the ribosomes of Escherichia coli. All the bridged macrolides investigated efficiently protected A2058 and A2059 in domain V of 23S rRNA from modification by dimethyl sulfate and U2609 from modification by carbodiimide. In addition, bridged macrolides that carry extended alkyl-aryl side chains protruding from the 6,11 bridge protected A752 in helix 35 of domain II of 23S rRNA from modification by dimethyl sulfate. Bridged macrolides efficiently displaced erythromycin from the ribosome in a competition binding assay. The A2058G mutation in 23S rRNA conferred resistance to the bridged macrolides. The U2609C mutation, which renders E. coli resistant to the previously studied ketolides telithromycin and cethromycin, barely affected cell susceptibility to the bridged macrolides used in this study. The results of the biochemical and genetic studies indicate that in the E. coli ribosome, bridged macrolides bind in the nascent peptide exit tunnel at the site previously described for other macrolide antibiotics. The presence of the side chain promotes the formation of specific interactions with the helix 35 of 23S rRNA.

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