Anomalous scattering analysis of Agrobacterium radiobacter phosphotriesterase: the prominent role of iron in the heterobinuclear active site

Bacterial phosphotriesterases are binuclear metalloproteins for which the catalytic mechanism has been studied with a variety of techniques, principally using active sites reconstituted in vitro from apoenzymes. Here, atomic absorption spectroscopy and anomalous X-ray scattering have been used to determine the identity of the metals incorporated into the active site in vivo. We have recombinantly expressed the phosphotriesterase from Agrobacterium radiobacter (OpdA) in Escherichia coli grown in medium supplemented with 1 mM CoCl2 and in unsupplemented medium. Anomalous scattering data, collected from a single crystal at the Fe–K, Co–K and Zn–K edges, indicate that iron and cobalt are the primary constituents of the two metal-binding sites in the catalytic centre (α and β) in the protein expressed in E. coli grown in supplemented medium. Comparison with OpdA expressed in unsupplemented medium demonstrates that the cobalt present in the supplemented medium replaced zinc at the β-position of the active site, which results in an increase in the catalytic efficiency of the enzyme. These results suggest an essential role for iron in the catalytic mechanism of bacterial phosphotriesterases, and that these phosphotriesterases are natively heterobinuclear iron–zinc enzymes.

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The Role of Glutathione in the Isomerization of Δ5-Androstene- 3,17-dione Catalyzed by Human Glutathione Transferase A1-1

Journal of Biological Chemistry ◽

10.1074/jbc.m009146200 ◽

2001 ◽

Vol 276 (15) ◽

pp. 11698-11704 ◽

Cited By ~ 37

Author(s):

Pär L. Pettersson ◽

Bengt Mannervik

Keyword(s):

Active Site ◽

Active Sites ◽

Catalytic Mechanism ◽

Glutathione Transferase ◽

Ph Dependence ◽

Enzymatic Catalysis ◽

Catalytic Efficiency ◽

Protonated Form ◽

Hydroxyl Group ◽

Isomerization Reaction

Human glutathione transferase (GST) A1-1 efficiently catalyzes the isomerization of Δ5-androstene-3,17-dione (AD) into Δ4-androstene-3,17-dione. High activity requires glutathione, but enzymatic catalysis occurs also in the absence of this cofactor. Glutathione alone shows a limited catalytic effect.S-Alkylglutathione derivatives do not promote the reaction, and the pH dependence of the isomerization indicates that the glutathione thiolate serves as a base in the catalytic mechanism. Mutation of the active-site Tyr9into Phe significantly decreases the steady-state kinetic parameters, alters their pH dependence, and increases the pKavalue of the enzyme-bound glutathione thiol. Thus, Tyr9promotes the reaction via its phenolic hydroxyl group in protonated form. GST A2-2 has a catalytic efficiency with AD 100-fold lower than the homologous GST A1-1. Another Alpha class enzyme, GST A4-4, is 1000-fold less active than GST A1-1. The Y9F mutant of GST A1-1 is more efficient than GST A2-2 and GST A4-4, both having a glutathione cofactor and an active-site Tyr9residue. The active sites of GST A2-2 and GST A1-1 differ by only four amino acid residues, suggesting that proper orientation of AD in relation to the thiolate of glutathione is crucial for high catalytic efficiency in the isomerization reaction. The GST A1-1-catalyzed steroid isomerization provides a complement to the previously described isomerase activity of 3β-hydroxysteroid dehydrogenase.

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Functional and computational identification of a rescue mutation near the active site of an mRNA methyltransferase

Scientific Reports ◽

10.1038/s41598-020-79026-2 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Pierre-Yves Colin ◽

Paul A. Dalby

Keyword(s):

Active Site ◽

Active Sites ◽

Large Scale ◽

Interaction Network ◽

Catalytic Efficiency ◽

Transferase Activity ◽

Viral Mrna ◽

In Vitro Production ◽

Cell Immunity

AbstractRNA-based drugs are an emerging class of therapeutics combining the immense potential of DNA gene-therapy with the absence of genome integration-associated risks. While the synthesis of such molecules is feasible, large scale in vitro production of humanised mRNA remains a biochemical and economical challenge. Human mRNAs possess two post-transcriptional modifications at their 5′ end: an inverted methylated guanosine and a unique 2′O-methylation on the ribose of the penultimate nucleotide. One strategy to precisely methylate the 2′ oxygen is to use viral mRNA methyltransferases that have evolved to escape the host’s cell immunity response following virus infection. However, these enzymes are ill-adapted to industrial processes and suffer from low turnovers. We have investigated the effects of homologous and orthologous active-site mutations on both stability and transferase activity, and identified new functional motifs in the interaction network surrounding the catalytic lysine. Our findings suggest that despite their low catalytic efficiency, the active-sites of viral mRNA methyltransferases have low mutational plasticity, while mutations in a defined third shell around the active site have strong effects on folding, stability and activity in the variant enzymes, mostly via network-mediated effects.

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Alternative catalytic residues in the active site of Esco acetyltransferases

10.1101/766543 ◽

2019 ◽

Author(s):

Tahereh Ajam ◽

Inessa De ◽

Nikolai Petkau ◽

Gabriela Whelan ◽

Vladimir Pena ◽

...

Keyword(s):

Active Site ◽

Catalytic Mechanism ◽

Mouse Embryonic Fibroblast ◽

Enzymatic Assays ◽

Sister Chromatids ◽

Proton Relay ◽

Active Site Cleft ◽

Embryonic Fibroblast

AbstractCohesin is a protein complex encircles the DNA and regulates the separation of sister chromatids during cell division. Following a catalytic mechanism that is insufficiently understood, Esco1 and Esco2 acetyltransferases acetylate Smc3 subunit of cohesin, thereby inducing a stabilization of cohesin on DNA. As a prerequisite for structure-guided investigation of enzymatic activity, we determine here the crystal structure of the mouse Esco2/CoA complex at 1.8 Å resolution. We reconstitute the entire cohesin as a tetrameric assembly and use it as a physiologically-relevant substrate for enzymatic assays in vitro. Furthermore, we employ cell-based complementation studies in mouse embryonic fibroblast deficient for Esco1 and Esco2, as a means to identify catalytically-important residues in vivo. These analyses demonstrate that D567/S566 and E491/S527, located on opposite sides of the MmEsco2 active site cleft, are critical for catalysis. Our experiments supports a catalytic mechanism of acetylation where residues D567 and E491 are general bases that deprotonate the ε-amino group of lysine substrate, via two nearby serine residues - S566 and S527-that possess a proton relay function.

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Functions of FKBP12 and Mitochondrial Cyclophilin Active Site Residues In Vitro and In Vivo inSaccharomyces cerevisiae

Molecular Biology of the Cell ◽

10.1091/mbc.8.11.2267 ◽

1997 ◽

Vol 8 (11) ◽

pp. 2267-2280 ◽

Cited By ~ 25

Author(s):

Kara Dolinski ◽

Christian Scholz ◽

R. Scott Muir ◽

Sabine Rospert ◽

Franz X. Schmid ◽

...

Keyword(s):

Active Site ◽

Active Sites ◽

In Vitro Assay ◽

Biologically Active ◽

Wild Type ◽

Prolyl Isomerase ◽

Vitro Assay ◽

Isomerase Activity

Cyclophilin and FK506 binding protein (FKBP) acceleratecis–trans peptidyl-prolyl isomerization and bind to and mediate the effects of the immunosuppressants cyclosporin A and FK506. The normal cellular functions of these proteins, however, are unknown. We altered the active sites of FKBP12 and mitochondrial cyclophilin from the yeast Saccharomyces cerevisiae by introducing mutations previously reported to inactivate these enzymes. Surprisingly, most of these mutant enzymes were biologically active in vivo. In accord with previous reports, all of the mutant enzymes had little or no detectable prolyl isomerase activity in the standard peptide substrate-chymotrypsin coupled in vitro assay. However, in a variation of this assay in which the protease is omitted, the mutant enzymes exhibited substantial levels of prolyl isomerase activity (5–20% of wild-type), revealing that these mutations confer sensitivity to protease digestion and that the classic in vitro assay for prolyl isomerase activity may be misleading. In addition, the mutant enzymes exhibited near wild-type activity with two protein substrates, dihydrofolate reductase and ribonuclease T1, whose folding is accelerated by prolyl isomerases. Thus, a number of cyclophilin and FKBP12 “active-site” mutants previously identified are largely active but protease sensitive, in accord with our findings that these mutants display wild-type functions in vivo. One mitochondrial cyclophilin mutant (R73A), and also the wild-type human FKBP12 enzyme, catalyze protein folding in vitro but lack biological activity in vivo in yeast. Our findings provide evidence that both prolyl isomerase activity and other structural features are linked to FKBP and cyclophilin in vivo functions and suggest caution in the use of these active-site mutations to study FKBP and cyclophilin functions.

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Endogenous Synthesis of Erythritol, a Novel Biomarker of Weight Gain (P15-016-19)

Current Developments in Nutrition ◽

10.1093/cdn/nzz037.p15-016-19 ◽

2019 ◽

Vol 3 (Supplement_1) ◽

Author(s):

Semira Ortiz ◽

Doletha Szebenyi ◽

Martha Field

Keyword(s):

Molecular Modeling ◽

Active Site ◽

Active Sites ◽

A549 Cells ◽

Site Directed Mutagenesis ◽

Central Adiposity ◽

Wild Type ◽

Reduction Activity

Abstract Objectives Serum erythritol is associated with central adiposity gain in young adults. Erythritol, a 4-carbon polyol, is synthesized endogenously from erythrose through the pentose phosphate pathway. We have identified two enzymes which catalyze this reaction: alcohol dehydrogenase 1 (ADH1) and sorbitol dehydrogenase (SORD). Interestingly, ADH1 isoforms ADH1B1 and ADH1C2 catalyze NADPH-dependent synthesis of erythritol in vitro, but ADH1B1 does not. In A549 cells, siRNA knockdown of SORD levels to less than 15% of control levels reduced erythritol by 50%. A549 cells also have low levels of ADH1 expression. This indicates that other enzymes may be capable of endogenous erythritol production. Based on its high degree of homology to ADH1, we hypothesize that ADH4 also catalyzes erythritol synthesis. The objective of this study was to further elucidate the mechanism of erythritol synthesis by: determining key differences between the active site of ADH1B2 compared to ADH1B1 and SORD, and screening a new candidate enzyme, ADH4. Methods This study used molecular modeling to evaluate the enzyme, erythrose, NADPH complex. Site-directed mutagenesis was performed to replace Arg208 (R208) of recombinant human SORD with histidine. Recombinant SORD R208H mutant and ADH4 were then expressed, purified, and their erythrose reduction activity was kinetically characterized. Results Molecular modeling of the active sites of ADH1B1 and ADH1B2 revealed that the Arg48 residue of ADH1B1 makes three contacts with NADPH, whereas His48 of ADH1B2 only makes one contact. Mutation of the SORD active-site Arg208 to histidine reduced the kcat by more than 80% compared to wild-type (95 ± 21 min−1 wild-type, 16 ± 2 min−1 R208H, P < 0.05). ADH4 erythrose reduction activity reduced by more than 95% compared to ADH1 variant proteins in vitro (kcat 2 ± 1 min−1). Conclusions Mutation of arginine 208 to histidine in the active site of SORD significantly reduced the kcat, indicating that the presence of an arginine residue is essential for binding NADPH, the cofactor required in vivo for erythritol synthesis. The low erythrose reduction activity of ADH4 suggests that ADH4 does not contribute to erythritol synthesis in humans. Funding Sources None.

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Probing the catalytically essential residues of a recombinant dipeptidyl carboxypeptidase from Escherichia coli

Biologia ◽

10.2478/s11756-010-0040-8 ◽

2010 ◽

Vol 65 (3) ◽

Cited By ~ 2

Author(s):

Huei-Fen Lo ◽

Hsiang-Ling Chen ◽

Shao-Yu Yen ◽

Ping-Lin Ong ◽

Wen-Shiue Chang ◽

...

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Active Site ◽

Metal Binding ◽

Catalytic Mechanism ◽

Zinc Ion ◽

Significant Loss ◽

In Vitro Mutagenesis ◽

Dipeptidyl Carboxypeptidase

AbstractIn studying the structure and function of Escherichia coli dipeptidyl carboxypeptidase (EcDCP), we have employed in vitro mutagenesis and subsequent protein expression to genetically dissect the enzyme in order to gain insight into the catalytic mechanism. Comparison of the amino acid sequence of EcDCP with other homologues indicates that the active site of the enzyme exhibits an HEXXH motif, a common feature of zinc metalloenzymes. The third metal binding ligand, presumed to coordinate directly to the active-site zinc ion in concert with His470 and His474 has been proposed as Glu499. Alterations to these residues completely abolished the catalytic activity against N-benzoyl-l-glycyl-l-histidyl-l-leucine. A significant loss of the enzymatic activity was also observed in F472V and F500V mutant enzymes. Intrinsic tryptophan fluorescence revealed the significant alterations of the microenvironment of aromatic amino acid residues in all mutant enzymes, whereas circular dichroism spectra were nearly identical for the tested proteins. Computer modeling suggests that residues His470, Glu471, His474, Glu499, and Phe500 are essential for EcDCP in maintaining the stable active-site environment. Taken together, these studies contribute to a more comprehensive understanding of the catalytic mechanism of the enzyme.

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Structures of c-di-GMP/cGAMP degrading phosphodiesterase VcEAL: identification of a novel conformational switch and its implication

Biochemical Journal ◽

10.1042/bcj20190399 ◽

2019 ◽

Vol 476 (21) ◽

pp. 3333-3353 ◽

Cited By ~ 3

Author(s):

Malti Yadav ◽

Kamalendu Pal ◽

Udayaditya Sen

Keyword(s):

Active Site ◽

Metal Binding ◽

Metal Ion ◽

Triosephosphate Isomerase ◽

Catalytic Mechanism ◽

Degradation Mechanism ◽

Structural Basis ◽

Conserved Residues ◽

Phosphodiester Bond ◽

Cyclic Dinucleotides

Cyclic dinucleotides (CDNs) have emerged as the central molecules that aid bacteria to adapt and thrive in changing environmental conditions. Therefore, tight regulation of intracellular CDN concentration by counteracting the action of dinucleotide cyclases and phosphodiesterases (PDEs) is critical. Here, we demonstrate that a putative stand-alone EAL domain PDE from Vibrio cholerae (VcEAL) is capable to degrade both the second messenger c-di-GMP and hybrid 3′3′-cyclic GMP–AMP (cGAMP). To unveil their degradation mechanism, we have determined high-resolution crystal structures of VcEAL with Ca2+, c-di-GMP-Ca2+, 5′-pGpG-Ca2+ and cGAMP-Ca2+, the latter provides the first structural basis of cGAMP hydrolysis. Structural studies reveal a typical triosephosphate isomerase barrel-fold with substrate c-di-GMP/cGAMP bound in an extended conformation. Highly conserved residues specifically bind the guanine base of c-di-GMP/cGAMP in the G2 site while the semi-conserved nature of residues at the G1 site could act as a specificity determinant. Two metal ions, co-ordinated with six stubbornly conserved residues and two non-bridging scissile phosphate oxygens of c-di-GMP/cGAMP, activate a water molecule for an in-line attack on the phosphodiester bond, supporting two-metal ion-based catalytic mechanism. PDE activity and biofilm assays of several prudently designed mutants collectively demonstrate that VcEAL active site is charge and size optimized. Intriguingly, in VcEAL-5′-pGpG-Ca2+ structure, β5–α5 loop adopts a novel conformation that along with conserved E131 creates a new metal-binding site. This novel conformation along with several subtle changes in the active site designate VcEAL-5′-pGpG-Ca2+ structure quite different from other 5′-pGpG bound structures reported earlier.

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Ancylostoma caninum Anticoagulant Peptide Blocks Metastasis In Vivo and Inhibits Factor Xa Binding to Melanoma Cells In Vitro

Thrombosis and Haemostasis ◽

10.1055/s-0037-1615117 ◽

1998 ◽

Vol 79 (05) ◽

pp. 1041-1047 ◽

Cited By ~ 20

Author(s):

Kathleen M. Donnelly ◽

Michael E. Bromberg ◽

Aaron Milstone ◽

Jennifer Madison McNiff ◽

Gordon Terwilliger ◽

...

Keyword(s):

Active Site ◽

Thrombin Generation ◽

Factor Xa ◽

Coagulation Factor ◽

Melanoma Cells ◽

Factor V ◽

Ancylostoma Caninum ◽

Metastatic Activity

SummaryWe evaluated the in vivo anti-metastatic activity of recombinant Ancylostoma caninum Anticoagulant Peptide (rAcAP), a potent (Ki = 265 pM) and specific active site inhibitor of human coagulation factor Xa originally isolated from bloodfeeding hookworms. Subcutaneous injection of SCID mice with rAcAP (0.01-0.2 mg/mouse) prior to tail vein injection of LOX human melanoma cells resulted in a dose dependent reduction in pulmonary metastases. In order to elucidate potential mechanisms of rAcAP’s anti-metastatic activity, experiments were carried out to identify specific interactions between factor Xa and LOX. Binding of biotinylated factor Xa to LOX monolayers was both specific and saturable (Kd = 15 nM). Competition experiments using antibodies to previously identified factor Xa binding proteins, including factor V/Va, effector cell protease receptor-1, and tissue factor pathway inhibitor failed to implicate any of these molecules as significant binding sites for Factor Xa. Functional prothrombinase activity was also supported by LOX, with a half maximal rate of thrombin generation detected at a factor Xa concentration of 2.4 nM. Additional competition experiments using an excess of either rAcAP or active site blocked factor Xa (EGR-Xa) revealed that most of the total factor Xa binding to LOX is mediated via interaction with the enzyme’s active site, predicting that the vast majority of cell-associated factor Xa does not participate directly in thrombin generation. In addition to establishing two distinct mechanisms of factor Xa binding to melanoma, these data raise the possibility that rAcAP’s antimetastatic effect in vivo might involve novel non-coagulant pathways, perhaps via inhibition of active-site mediated interactions between factor Xa and tumor cells.

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Action of Thioglycosides of 1,2,4-Triazoles and Imidazoles on the Oxidative Stress and Glycosidases in Mice with Molecular Docking

Letters in Drug Design & Discovery ◽

10.2174/1573413715666181212150955 ◽

2019 ◽

Vol 16 (6) ◽

pp. 696-710

Author(s):

Mahmoud Balbaa ◽

Doaa Awad ◽

Ahmad Abd Elaal ◽

Shimaa Mahsoub ◽

Mayssaa Moharram ◽

...

Keyword(s):

Oxidative Stress ◽

Molecular Docking ◽

Active Sites ◽

Biological Activities ◽

Imidazole Ring ◽

Quantitative Structure Activity Relationship ◽

Binding Interaction ◽

Qsar Study

Background: ,2,3-Triazoles and imidazoles are important five-membered heterocyclic scaffolds due to their extensive biological activities. These products have been an area of growing interest to many researchers around the world because of their enormous pharmaceutical scope. Methods: The in vivo and in vitro enzyme inhibition of some thioglycosides encompassing 1,2,4- triazole N1, N2, and N3 and/or imidazole moieties N4, N5, and N6. The effect on the antioxidant enzymes (superoxide dismutase, glutathione S-transferase, glutathione peroxidase and catalase) was investigated as well as their effect on α-glucosidase and β-glucuronidase. Molecular docking studies were carried out to investigate the mode of the binding interaction of the compounds with α- glucosidase and β -glucuronidase. In addition, quantitative structure-activity relationship (QSAR) investigation was applied to find out the correlation between toxicity and physicochemical properties. Results: The decrease of the antioxidant status was revealed by the in vivo effect of the tested compounds. Furthermore, the in vivo and in vitro inhibitory effects of the tested compounds were clearly pronounced on α-glucosidase, but not β-glucuronidase. The IC50 and Ki values revealed that the thioglycoside - based 1,2,4-triazole N3 possesses a high inhibitory action. In addition, the in vitro studies demonstrated that the whole tested 1,2,4-triazole are potent inhibitors with a Ki magnitude of 10-6 and exhibited a competitive type inhibition. On the other hand, the thioglycosides - based imidazole ring showed an antioxidant activity and exerted a slight in vivo stimulation of α-glucosidase and β- glucuronidase. Molecular docking proved that the compounds exhibited binding affinity with the active sites of α -glucosidase and β-glucuronidase (docking score ranged from -2.320 to -4.370 kcal/mol). Furthermore, QSAR study revealed that the HBD and RB were found to have an overall significant correlation with the toxicity. Conclusion: These data suggest that the inhibition of α-glucosidase is accompanied by an oxidative stress action.

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Functional dissection of the catalytic mechanism of mammalian RNA polymerase II

Biochemistry and Cell Biology ◽

10.1139/o05-061 ◽

2005 ◽

Vol 83 (4) ◽

pp. 497-504 ◽

Cited By ~ 2

Author(s):

Benoit Coulombe ◽

Marie-France Langelier

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Catalytic Mechanism ◽

Mutational Analysis ◽

Structural Elements ◽

Catalytic Mechanisms ◽

Rnap Ii ◽

Affinity Peptide

High resolution X-ray crystal structures of multisubunit RNA polymerases (RNAP) have contributed to our understanding of transcriptional mechanisms. They also provided a powerful guide for the design of experiments aimed at further characterizing the molecular stages of the transcription reaction. Our laboratory used tandem-affinity peptide purification in native conditions to isolate human RNAP II variants that had site-specific mutations in structural elements located strategically within the enzyme's catalytic center. Both in vitro and in vivo analyses of these mutants revealed novel features of the catalytic mechanisms involving this enzyme.Key words: RNA polymerase II, transcriptional mechanisms, mutational analysis, mRNA synthesis.

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