Alternative catalytic residues in the active site of Esco acetyltransferases

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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Phytochemical Characterization and Chemotherapeutic Potential of Cinnamomum verum Extracts on the Multiplication of Protozoan Parasites In Vitro and In Vivo

Molecules ◽

10.3390/molecules25040996 ◽

2020 ◽

Vol 25 (4) ◽

pp. 996 ◽

Cited By ~ 8

Author(s):

Gaber El-Saber Batiha ◽

Amany Magdy Beshbishy ◽

Azirwan Guswanto ◽

Arifin Nugraha ◽

Tserendorj Munkhjargal ◽

...

Keyword(s):

Drug Exposure ◽

Inhibitory Effect ◽

Mouse Embryonic Fibroblast ◽

Bioactive Constituents ◽

Viability Assay ◽

Nih 3T3 ◽

Embryonic Fibroblast ◽

Herbal Plant

Cinnamomum verum is a commonly used herbal plant that has several documented properties against various diseases. The existing study evaluated the inhibitory effect of acetonic extract of C. verum (AECV) and ethyl acetate extract of C. verum (EAECV) against piroplasm parasites in vitro and in vivo. The drug-exposure viability assay was tested on Madin-Darby bovine kidney (MDBK), mouse embryonic fibroblast (NIH/3T3) and human foreskin fibroblast (HFF) cells. Qualitative phytochemical estimation revealed that AECV and EAECV containing multiple bioactive constituents namely alkaloids, tannins, saponins, terpenoids and remarkable amounts of polyphenols and flavonoids. AECV and EAECV inhibited B. bovis, B. bigemina, B. divergens, B. caballi, and T. equi multiplication at half-maximal inhibitory concentrations (IC50) of 23.1 ± 1.4, 56.6 ± 9.1, 33.4 ± 2.1, 40.3 ± 7.5, 18.8 ± 1.6 µg/mL, and 40.1 ± 8.5, 55.6 ± 1.1, 45.7 ± 1.9, 50.2 ± 6.2, and 61.5 ± 5.2 µg/mL, respectively. In the cytotoxicity assay, AECV and EAECV affected the viability of MDBK, NIH/3T3 and HFF cells with half-maximum effective concentrations (EC50) of 440 ± 10.6, 816 ± 12.7 and 914 ± 12.2 µg/mL and 376 ± 11.2, 610 ± 7.7 and 790 ± 12.4 µg/mL, respectively. The in vivo experiment showed that AECV and EAECV were effective against B. microti in mice at 150 mg/kg. These results showed that C. verum extracts are potential antipiroplasm drugs after further studies in some clinical cases.

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Therapeutic Effects of Atranorin towards the Proliferation of Babesia and Theileria Parasites

Pathogens ◽

10.3390/pathogens9020127 ◽

2020 ◽

Vol 9 (2) ◽

pp. 127 ◽

Cited By ~ 11

Author(s):

Amany Magdy Beshbishy ◽

Gaber El-Saber Batiha ◽

Luay Alkazmi ◽

Eman Nadwa ◽

Eman Rashwan ◽

...

Keyword(s):

Mouse Embryonic Fibroblast ◽

Drug Candidate ◽

Therapeutic Effects ◽

Diminazene Aceturate ◽

Human Foreskin Fibroblasts ◽

Ic50 Values ◽

Nih 3T3 ◽

Embryonic Fibroblast

Atranorin (ATR), is a compound with multidirectional biological activity under different in vitro and in vivo conditions and it is effective as an antibacterial, antiviral, antiprotozoal and anti-inflammatory agent. In the current study, the in vitro as well as in vivo chemotherapeutic effect of ATR as well as its combined efficacy with the existing antibabesial drugs (diminazene aceturate (DA), atovaquone (AV) and clofazimine (CF)) were investigated on six species of piroplasm parasites. ATR suppressed B. bovis, B. bigemina, B. divergens, B. caballi and T. equi multiplication in vitro with IC50 values of 98.4 ± 4.2, 64.5 ± 3.9, 45.2 ± 5.9, 46.6 ± 2.5, and 71.3 ± 2.7 µM, respectively. The CCK test was used to examine ATR’s cytotoxicity and adverse effects on different animal and human cell lines, the main hosts of piroplasm parasites and it showed that ATR affected human foreskin fibroblasts (HFF), mouse embryonic fibroblast (NIH/3T3) and Madin-Darby Bovine Kidney (MDBK) cell viability in a dose-related effect with a moderate selective index. The combined efficacy of ATR with DA, CF, and AV exhibited a synergistic and additive efficacy toward all tested species. In the in vivo experiment, ATR prohibited B. microti multiplication in mice by 68.17%. The ATR-DA and ATR-AV combination chemotherapies were more potent than ATR monotherapy. These results indicate the prospects of ATR as a drug candidate for piroplasmosis treatment.

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

Biochemical Journal ◽

10.1042/bj20060276 ◽

2006 ◽

Vol 397 (3) ◽

pp. 501-508 ◽

Cited By ~ 46

Author(s):

Colin J. Jackson ◽

Paul D. Carr ◽

Hye-Kyung Kim ◽

Jian-Wei Liu ◽

Paul Herrald ◽

...

Keyword(s):

Active Site ◽

Metal Binding ◽

Active Sites ◽

Catalytic Mechanism ◽

Catalytic Efficiency ◽

Scattering Data ◽

Anomalous Scattering ◽

Agrobacterium Radiobacter

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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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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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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Important Role for Phylogenetically Invariant PP2Acα Active Site and C-Terminal Residues Revealed by Mutational Analysis in Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/156.1.21 ◽

2000 ◽

Vol 156 (1) ◽

pp. 21-29 ◽

Cited By ~ 1

Author(s):

David R H Evans ◽

Brian A Hemmings

Keyword(s):

Protein Interactions ◽

Active Site ◽

Protein Function ◽

Mutational Analysis ◽

Yeast Cells ◽

Temperature Sensitive ◽

Active Site Residues ◽

Catalytic Function

Abstract PP2A is a central regulator of eukaryotic signal transduction. The human catalytic subunit PP2Acα functionally replaces the endogenous yeast enzyme, Pph22p, indicating a conservation of function in vivo. Therefore, yeast cells were employed to explore the role of invariant PP2Ac residues. The PP2Acα Y127N substitution abolished essential PP2Ac function in vivo and impaired catalysis severely in vitro, consistent with the prediction from structural studies that Tyr-127 mediates substrate binding and its side chain interacts with the key active site residues His-118 and Asp-88. The V159E substitution similarly impaired PP2Acα catalysis profoundly and may cause global disruption of the active site. Two conditional mutations in the yeast Pph22p protein, F232S and P240H, were found to cause temperature-sensitive impairment of PP2Ac catalytic function in vitro. Thus, the mitotic and cell lysis defects conferred by these mutations result from a loss of PP2Ac enzyme activity. Substitution of the PP2Acα C-terminal Tyr-307 residue by phenylalanine impaired protein function, whereas the Y307D and T304D substitutions abolished essential function in vivo. Nevertheless, Y307D did not reduce PP2Acα catalytic activity significantly in vitro, consistent with an important role for the C terminus in mediating essential protein-protein interactions. Our results identify key residues important for PP2Ac function and characterize new reagents for the study of PP2A in vivo.

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ZN148 Is a Modular Synthetic Metallo-β-Lactamase Inhibitor That Reverses Carbapenem Resistance in Gram-Negative Pathogens In Vivo

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02415-19 ◽

2020 ◽

Vol 64 (6) ◽

Cited By ~ 3

Author(s):

Ørjan Samuelsen ◽

Ove Alexander Høgmoen Åstrand ◽

Christopher Fröhlich ◽

Adam Heikal ◽

Susann Skagseth ◽

...

Keyword(s):

Active Site ◽

Therapeutic Potential ◽

Treatment Options ◽

Carbapenem Resistance ◽

Functional Space ◽

Bactericidal Effect ◽

Gram Negative ◽

Content Type

ABSTRACT Carbapenem-resistant Gram-negative pathogens are a critical public health threat and there is an urgent need for new treatments. Carbapenemases (β-lactamases able to inactivate carbapenems) have been identified in both serine β-lactamase (SBL) and metallo-β-lactamase (MBL) families. The recent introduction of SBL carbapenemase inhibitors has provided alternative therapeutic options. Unfortunately, there are no approved inhibitors of MBL-mediated carbapenem-resistance and treatment options for infections caused by MBL-producing Gram-negatives are limited. Here, we present ZN148, a zinc-chelating MBL-inhibitor capable of restoring the bactericidal effect of meropenem and in vitro clinical susceptibility to carbapenems in >98% of a large international collection of MBL-producing clinical Enterobacterales strains (n = 234). Moreover, ZN148 was able to potentiate the effect of meropenem against NDM-1-producing Klebsiella pneumoniae in a murine neutropenic peritonitis model. ZN148 showed no inhibition of the human zinc-containing enzyme glyoxylase II at 500 μM, and no acute toxicity was observed in an in vivo mouse model with cumulative dosages up to 128 mg/kg. Biochemical analysis showed a time-dependent inhibition of MBLs by ZN148 and removal of zinc ions from the active site. Addition of exogenous zinc after ZN148 exposure only restored MBL activity by ∼30%, suggesting an irreversible mechanism of inhibition. Mass-spectrometry and molecular modeling indicated potential oxidation of the active site Cys221 residue. Overall, these results demonstrate the therapeutic potential of a ZN148-carbapenem combination against MBL-producing Gram-negative pathogens and that ZN148 is a highly promising MBL inhibitor that is capable of operating in a functional space not presently filled by any clinically approved compound.

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Construction of adenovirus vectors simultaneously expressing four multiplex, double-nicking guide RNAs of CRISPR/Cas9 and in vivo genome editing

Scientific Reports ◽

10.1038/s41598-021-83259-0 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Tomoko Nakanishi ◽

Aya Maekawa ◽

Mariko Suzuki ◽

Hirotaka Tabata ◽

Kumiko Sato ◽

...

Keyword(s):

Target Gene ◽

Animal Experiments ◽

Adenovirus Vector ◽

Mouse Embryonic Fibroblast ◽

Newborn Mouse ◽

Guide Rnas ◽

One Step ◽

Target Effect

AbstractSimultaneous expression of multiplex guide RNAs (gRNAs) is valuable for knockout of multiple genes and also for effective disruption of a gene by introducing multiple deletions. We developed a method of Tetraplex-guide Tandem for construction of cosmids containing four and eight multiplex gRNA-expressing units in one step utilizing lambda in vitro packaging. Using this method, we produced an adenovirus vector (AdV) containing four multiplex-gRNA units for two double-nicking sets. Unexpectedly, the AdV could stably be amplified to the scale sufficient for animal experiments with no detectable lack of the multiplex units. When the AdV containing gRNAs targeting the H2-Aa gene and an AdV expressing Cas9 nickase were mixed and doubly infected to mouse embryonic fibroblast cells, deletions were observed in more than 80% of the target gene even using double-nicking strategy. Indels were also detected in about 20% of the target gene at two sites in newborn mouse liver cells by intravenous injection. Interestingly, when one double-nicking site was disrupted, the other was simultaneously disrupted, implying that two genes in the same cell may simultaneously be disrupted in the AdV system. The AdVs expressing four multiplex gRNAs could offer simultaneous knockout of four genes or two genes by double-nicking cleavages with low off-target effect.

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ADP-Ribose-1"-Monophosphatase: a Conserved Coronavirus Enzyme That Is Dispensable for Viral Replication in Tissue Culture

Journal of Virology ◽

10.1128/jvi.79.20.12721-12731.2005 ◽

2005 ◽

Vol 79 (20) ◽

pp. 12721-12731 ◽

Cited By ~ 99

Author(s):

Ákos Putics ◽

Witold Filipowicz ◽

Jonathan Hall ◽

Alexander E. Gorbalenya ◽

John Ziebuhr

Keyword(s):

Active Site ◽

Biological Significance ◽

Virus Titer ◽

Site Directed Mutagenesis ◽

Replicase Gene ◽

Active Site Residues ◽

Nonstructural Proteins ◽

Trna Splicing

ABSTRACT Replication of the ∼30-kb plus-strand RNA genome of coronaviruses and synthesis of an extensive set of subgenome-length RNAs are mediated by the replicase-transcriptase, a membrane-bound protein complex containing several cellular proteins and up to 16 viral nonstructural proteins (nsps) with multiple enzymatic activities, including protease, polymerase, helicase, methyltransferase, and RNase activities. To get further insight into the replicase gene-encoded functions, we characterized the coronavirus X domain, which is part of nsp3 and has been predicted to be an ADP-ribose-1"-monophosphate (Appr-1"-p) processing enzyme. Bacterially expressed forms of human coronavirus 229E (HCoV-229E) and severe acute respiratory syndrome-coronavirus X domains were shown to dephosphorylate Appr-1"-p, a side product of cellular tRNA splicing, to ADP-ribose in a highly specific manner. The enzyme had no detectable activity on several other nucleoside phosphates. Guided by the crystal structure of AF1521, an X domain homolog from Archaeoglobus fulgidus, potential active-site residues of the HCoV-229E X domain were targeted by site-directed mutagenesis. The data suggest that the HCoV-229E replicase polyprotein residues, Asn 1302, Asn 1305, His 1310, Gly 1312, and Gly 1313, are part of the enzyme's active site. Characterization of an Appr-1"-pase-deficient HCoV-229E mutant revealed no significant effects on viral RNA synthesis and virus titer, and no reversion to the wild-type sequence was observed when the mutant virus was passaged in cell culture. The apparent dispensability of the conserved X domain activity in vitro indicates that coronavirus replicase polyproteins have evolved to include nonessential functions. The biological significance of the novel enzymatic activity in vivo remains to be investigated.

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