scholarly journals Interaction of the Sliding Clamp β-Subunit and Hda, a DnaA-Related Protein

2004 ◽  
Vol 186 (11) ◽  
pp. 3508-3515 ◽  
Author(s):  
Mareike Kurz ◽  
Brian Dalrymple ◽  
Gene Wijffels ◽  
Kritaya Kongsuwan
Keyword(s):  
Amino Acid ◽  
Consensus Sequence ◽  
Nitrilotriacetic Acid ◽  
Replication Initiation ◽  
Binding Motif ◽  
Β Subunit ◽  
Related Protein ◽  
Sliding Clamp ◽  
Hydrolysis Of

ABSTRACT In Escherichia coli, interactions between the replication initiation protein DnaA, the β subunit of DNA polymerase III (the sliding clamp protein), and Hda, the recently identified DnaA-related protein, are required to convert the active ATP-bound form of DnaA to an inactive ADP-bound form through the accelerated hydrolysis of ATP. This rapid hydrolysis of ATP is proposed to be the main mechanism that blocks multiple initiations during cell cycle and acts as a molecular switch from initiation to replication. However, the biochemical mechanism for this crucial step in DNA synthesis has not been resolved. Using purified Hda and β proteins in a plate binding assay and Ni-nitrilotriacetic acid pulldown analysis, we show for the first time that Hda directly interacts with β in vitro. A new β-binding motif, a hexapeptide with the consensus sequence QL[SP]LPL, related to the previously identified β-binding pentapeptide motif (QL[SD]LF) was found in the amino terminus of the Hda protein. Mutants of Hda with amino acid changes in the hexapeptide motif are severely defective in their ability to bind β. A 10-amino-acid peptide containing the E. coli Hda β-binding motif was shown to compete with Hda for binding to β in an Hda-β interaction assay. These results establish that the interaction of Hda with β is mediated through the hexapeptide sequence. We propose that this interaction may be crucial to the events that lead to the inactivation of DnaA and the prevention of excess initiation of rounds of replication.

scholarly journals ywfE in Bacillus subtilis Codes for a Novel Enzyme, l-Amino Acid Ligase

2005 ◽  
Vol 187 (15) ◽  
pp. 5195-5202 ◽  
Author(s):  
Kazuhiko Tabata ◽  
Hajime Ikeda ◽  
Shin-ichi Hashimoto
Keyword(s):  
Amino Acids ◽  
Bacillus Subtilis ◽  
Amino Acid ◽  
High Performance ◽  
Consensus Sequence ◽  
Reaction Products ◽  
In Silico Screening ◽  
Vitro Assay ◽  
Hydrolysis Of

ABSTRACT The ATP-dependent carboxylate-amine/thiol ligase superfamily is known to contain enzymes catalyzing the formation of various types of peptide, such as d-alanyl-d-alanine, polyglutamate, and γ-peptide, but, curiously, no enzyme synthesizing α-dipeptides of l-amino acids is known. We attempted to find such an enzyme. By in silico screening based on the consensus sequence of the superfamily followed by an in vitro assay with purified enzyme to avoid the degradation of the peptide(s) synthesized, ywfE of Bacillus subtilis was found to code for the activity forming l-alanyl-l-glutamine from l-alanine and l-glutamine with hydrolysis of ATP to ADP. No AMP was formed, supporting the idea that the enzyme belongs to the superfamily. Surprisingly, the enzyme accepted a wide variety of l-amino acids. Among 231 combinations of l-amino acids tested, reaction products were obtained for 111 combinations and 44 kinds of α-dipeptides were confirmed by high-performance liquid chromatography analyses, while no tripeptide or longer peptide was detected and the d-amino acids were inert. From these results, we propose that ywfE encodes a new member of the superfamily, l-amino acid ligase.

scholarly journals Novel Amino Acid Derivatives of Quinolines as Potential Antibacterial and Fluorophore Agents

2020 ◽  
Vol 88 (4) ◽  
pp. 57
Author(s):  
Oussama Moussaoui ◽  
Rajendra Bhadane ◽  
Riham Sghyar ◽  
El Mestafa El Hadrami ◽  
Soukaina El Amrani ◽  
...  
Keyword(s):  
Amino Acid ◽  
Methyl Esters ◽  
Amino Acid Derivatives ◽  
Bacterial Strains ◽  
Fluorescent Properties ◽  
Topoisomerase Iv ◽  
Microdilution Method ◽  
Hydrolysis Of ◽  
Derivatives Of

A new series of amino acid derivatives of quinolines was synthesized through the hydrolysis of amino acid methyl esters of quinoline carboxamides with alkali hydroxide. The compounds were purified on silica gel by column chromatography and further characterized by TLC, NMR and ESI-TOF mass spectrometry. All compounds were screened for in vitro antimicrobial activity against different bacterial strains using the microdilution method. Most of the synthesized amino acid-quinolines show more potent or equipotent inhibitory action against the tested bacteria than their correspond esters. In addition, many of them exhibit fluorescent properties and could possibly be utilized as fluorophores. Molecular docking and simulation studies of the compounds at putative bacterial target enzymes suggest that the antimicrobial potency of these synthesized analogues could be due to enzyme inhibition via their favorable binding at the fluoroquinolone binding site at the GyrA subunit of DNA gyrase and/or the ParC subunit of topoisomerase-IV.

scholarly journals Attenuation of Murray Valley Encephalitis Virus by Site-Directed Mutagenesis of the Hinge and Putative Receptor-Binding Regions of the Envelope Protein

2001 ◽  
Vol 75 (16) ◽  
pp. 7692-7702 ◽  
Author(s):  
Robert J. Hurrelbrink ◽  
Peter C. McMinn
Keyword(s):  
Amino Acid ◽  
Receptor Binding ◽  
Encephalitis Virus ◽  
Hinge Region ◽  
Wild Type Virus ◽  
Binding Motif ◽  
Murray Valley Encephalitis Virus ◽  
Ligand Interaction

ABSTRACT Molecular determinants of virulence in flaviviruses cluster in two regions on the three-dimensional structure of the envelope (E) protein; the base of domain II, believed to serve as a hinge during pH-dependent conformational change in the endosome, and the lateral face of domain III, which contains an integrin-binding motif Arg-Gly-Asp (RGD) in mosquito-borne flaviviruses and is believed to form the receptor-binding site of the protein. In an effort to better understand the nature of attenuation caused by mutations in these two regions, a full-length infectious cDNA clone of Murray Valley encephalitis virus prototype strain 1-51 (MVE-1-51) was employed to produce a panel of site-directed mutants with substitutions at amino acid positions 277 (E-277; hinge region) or 390 (E-390; RGD motif). Viruses with mutations at E-277 (Ser→Ile, Ser→Asn, Ser→Val, and Ser→Pro) showed various levels of in vitro and in vivo attenuation dependent on the level of hydrophobicity of the substituted amino acid. Altered hemagglutination activity observed for these viruses suggests that mutations in the hinge region may indirectly disrupt the receptor-ligand interaction, possibly by causing premature release of the virion from the endosomal membrane prior to fusion. Similarly, viruses with mutations at E-390 (Asp→Asn, Asp→Glu, and Asp→Tyr) were also attenuated in vitro and in vivo; however, the absorption and penetration rates of these viruses were similar to those of wild-type virus. This, coupled with the fact that E-390 mutant viruses were only moderately inhibited by soluble heparin, suggests that RGD-dependent integrin binding is not essential for entry of MVE and that multiple and/or alternate receptors may be involved in cell entry.

scholarly journals C-terminal tripeptide Ser-Asn-Leu (SNL) of human D-aspartate oxidase is a functional peroxisome-targeting signal

1998 ◽  
Vol 336 (2) ◽  
pp. 367-371 ◽  
Author(s):  
Leen AMERY ◽  
Chantal BREES ◽  
Myriam BAES ◽  
Chiaki SETOYAMA ◽  
Retsu MIURA ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Amino Acid ◽  
Fluorescent Protein ◽  
Consensus Sequence ◽  
C Terminus ◽  
Targeting Signal ◽  
Fluorescent Pattern ◽  
Green Fluorescent ◽  
Positively Charged

The functionality of the C-terminus (Ser-Asn-Leu; SNL) of human d-aspartate oxidase, an enzyme proposed to have a role in the inactivation of synaptically released d-aspartate, as a peroxisome-targeting signal (PTS1) was investigated in vivoand in vitro. Bacterially expressed human d-aspartate oxidase was shown to interact with the human PTS1-binding protein, peroxin protein 5 (PEX5p). Binding was gradually abolished by carboxypeptidase treatment of the oxidase and competitively inhibited by a Ser-Lys-Leu (SKL)-containing peptide. After transfection of mouse fibroblasts with a plasmid encoding green fluorescent protein (GFP) extended by PKSNL (the C-terminal pentapeptide of the oxidase), a punctate fluorescent pattern was evident. The modified GFP co-localized with peroxisomal thiolase as shown by indirect immunofluorescence. On transfection in fibroblasts lacking PEX5p receptor, GFP–PKSNL staining was cytosolic. Peroxisomal import of GFP extended by PGSNL (replacement of the positively charged fourth-last amino acid by glycine) seemed to be slower than that of GFP–PKSNL, whereas extension by PKSNG abolished the import of the modified GFP. Taken together, these results indicate that SNL, a tripeptide not fitting the PTS1 consensus currently defined in mammalian systems, acts as a functional PTS1 in mammalian systems, and that the consensus sequence, based on this work and that of other groups, has to be broadened to (S/A/C/K/N)-(K/R/H/Q/N/S)-L.

scholarly journals Respiratory Syncytial Virus M2-1 Protein Requires Phosphorylation for Efficient Function and Binds Viral RNA during Infection

2001 ◽  
Vol 75 (24) ◽  
pp. 12188-12197 ◽  
Author(s):  
Tara L. Cartee ◽  
Gail W. Wertz
Keyword(s):  
Protein Function ◽  
Consensus Sequence ◽  
Casein Kinase ◽  
Rnase A ◽  
Site Directed Mutagenesis ◽  
Binding Motif ◽  
Casein Kinase I ◽  
N Protein ◽  
Syncytial Virus

ABSTRACT The M2-1 protein of respiratory syncytial (RS) virus is a transcriptional processivity and antitermination factor. The M2-1 protein has a Cys3His1 zinc binding motif which is essential for function, is phosphorylated, and has been shown to interact with the RS virus nucleocapsid (N) protein. In the work reported here, we determined the sites at which the M2-1 protein was phosphorylated and investigated the importance of these phosphorylated residues for M2-1 function in transcription. By combining protease digestion, matrix-assisted laser desorption ionization–time of flight mass spectrometry, and site-directed mutagenesis, we identified the phosphorylated residues as serines 58 and 61, not threonine 56 and serine 58 as previously reported. Serines 58 and 61 and the surrounding amino acids are in a consensus sequence for phosphorylation by casein kinase I. Consistent with this, we showed that the unphosphorylated M2-1 protein synthesized in Escherichia coli could be phosphorylated in vitro by casein kinase I. The effect of eliminating phosphorylation by site-specific mutagenesis of serines 58 and 61 on the function of the M2-1 protein in transcription of RS virus subgenomic replicons was assayed. The activities of the M2-1 protein phosphorylation mutants in transcriptional antitermination were tested over a range of concentrations and were found to be substantially inhibited at all concentrations. The data show that phosphorylation is important for the M2-1 protein function in transcription. However, mutation of the M2-1 phosphorylation sites did not interfere with the ability of the M2-1 protein to interact with the N protein in transfected cells. The interaction of the M2-1 and N proteins in cotransfected cells was found to be sensitive to RNase A, indicating that the M2-1–N protein interaction was mediated via RNA. Furthermore, the M2-1 protein was shown to bind monocistronic and polycistronic RS virus mRNAs during infection.

scholarly journals cDNAs encoding the large subunit of human replication factor C

1993 ◽  
Vol 90 (23) ◽  
pp. 11014-11018 ◽  
Author(s):  
F Bunz ◽  
R Kobayashi ◽  
B Stillman
Keyword(s):  
Amino Acid ◽  
Large Subunit ◽  
Simian Virus 40 ◽  
Amino Acid Sequences ◽  
Binding Motif ◽  
Replication Factor C ◽  
Viral Origin ◽  
Replication Factor ◽  
Factor C

Replication factor C (RFC) is a multisubunit, DNA polymerase accessory protein required for the coordinated synthesis of both DNA strands during simian virus 40 DNA replication in vitro. Previous studies have shown that RFC is a DNA-dependent ATPase that binds in a structure-specific manner to the 3' end of a primer hybridized to a template DNA, an activity thought intrinsic to the 140-kDa component of this multisubunit complex. Here, the isolation and analysis of cDNAs encoding this subunit is described. Analysis of the full-length coding sequence revealed an open reading frame of 3.4 kb, encoding an 1148-amino acid protein with a predicted molecular mass of 130 kDa. A putative ATP-binding motif was observed that is similar to a motif in several of the smaller subunits of RFC and in functionally homologous replication factors of bacterial and viral origin. A "DEAD" box is also conserved among these proteins. The predicted protein shows significant identity with a DNA-binding protein of murine origin (B. Luckow, P. Lichter, and G. Schütz, personal communication). Regions of similarity were also seen between the amino acid sequences of the 140-kDa subunit of RFC, poly(ADP-ribose) polymerase, and bacterial DNA ligases--possibly representing a conserved structural feature of these proteins that bind similar DNA substrates.

scholarly journals Yeast Rgd3 is a phospho-regulated F-BAR-containing RhoGAP involved in the regulation of Rho3 distribution and cell morphology

2020 ◽  
Author(s):  
Robert M. Gingras ◽  
Kyaw Myo Lwin ◽  
Abigail M. Miller ◽  
Anthony Bretscher
Keyword(s):  
Cell Polarity ◽  
Binding Motif ◽  
Restrictive Temperature ◽  
Secretory Vesicles ◽  
Polarized Growth ◽  
Related Protein ◽  
Myosin V ◽  
Over Expression ◽  
Dependent Transport

AbstractPolarized growth requires the integration of polarity pathways with the delivery of exocytic vesicles for cell expansion and counterbalancing endocytic uptake. In budding yeast, the myosin-V Myo2 is aided by the kinesin-related protein Smy1 in carrying out the essential Sec4-dependent transport of secretory vesicles to sites of polarized growth. Over-expression suppressors of a conditional myo2 smy1 mutant identified a novel F-BAR-containing RhoGAP, Rgd3, that has activity primarily on Rho3, but also Cdc42. Internally tagged Rho3 is restricted to the plasma membrane in a gradient corresponding to cell polarity that is altered upon Rgd3 over-expression. Rgd3 itself is localized to dynamic polarized vesicles that, while distinct from constitutive secretory vesicles, are dependent on actin and Myo2 function. In vitro Rgd3 associates with liposomes in a PIP2-enhanced manner. Further, the Rgd3 C-terminal region contains several phosphorylatable residues within a reported SH3-binding motif. An unphosphorylated mimetic construct is active and highly polarized, while the phospho-mimetic form is not. Rgd3 is capable of activating Myo2, dependent on its phospho-state and Rgd3 overexpression rescues aberrant Rho3 localization and cell morphologies seen at the restrictive temperature in the myo2 smy1 mutant. We propose a model where Rgd3 functions to modulate and maintain Rho3 polarity during growth.

scholarly journals Amino Acid, Mineral, and Degree of Hydrolysis of Vinegar-Egg and Its Lipid Lowering and Antioxidant Effects in vitro and in vivo

2019 ◽  
Vol 48 (8) ◽  
pp. 1643-1654
Author(s):  
Yue Zheng ◽  
Kun Liu ◽  
Wanghui Yan ◽  
Guohua Wei ◽  
Xinyu Chao ◽  
...  
Keyword(s):  
Amino Acid ◽  
Lipid Lowering ◽  
Degree Of Hydrolysis ◽  
Hydrolysis Of ◽  
Antioxidant Effects

scholarly journals Selection of the same mutation in the U69 protein kinase gene of human herpesvirus-6 after prolonged exposure to ganciclovir in vitro and in vivo

2001 ◽  
Vol 82 (11) ◽  
pp. 2767-2776 ◽  
Author(s):  
Chaysavanh Manichanh ◽  
Camille Olivier-Aubron ◽  
Jean-Pierre Lagarde ◽  
Jean-Thierry Aubin ◽  
Phillipe Bossi ◽  
...  
Keyword(s):  
Amino Acid ◽  
Protein Kinase ◽  
Amino Acid Substitution ◽  
Consensus Sequence ◽  
Human Herpesvirus ◽  
Wild Type ◽  
Human Herpesvirus 6 ◽  
Genotypic Analysis ◽  
Herpesvirus 6

After serial passage in the presence of increasing concentrations of ganciclovir (GCV) in vitro, a human herpesvirus-6 (HHV-6) mutant exhibiting a decreased sensitivity to the drug was isolated. Analysis of drug susceptibility showed that the IC50 of this mutant was 24-, 52- and 3-fold higher than that of the wild-type (wt) IC50 in the case of GCV, cidofovir and foscarnet, respectively. Genotypic analysis showed two single nucleotide changes as compared to the wild-type: an A→G substitution of the U69 protein kinase (PK) gene resulted in an M318V amino acid substitution and the other change, located in the C-terminal part of the U38 gene, resulted in an A961V amino acid substitution within the DNA polymerase. The M318V change was located within the consensus sequence DISPMN of the putative catalytic domain VI of the PK. This change was homologous to the M460V and M460I changes that had been reported previously within the consensus sequence DITPMN of the human cytomegalovirus (HCMV) UL97 PK and associated with the resistance of HCMV to GCV. The M318V change was also detected by PCR in HHV-6-infected PBMCs from an AIDS patient who had been treated with GCV for a long period of time and exhibited a clinically GCV-resistant HCMV infection. These findings provide strong circumstantial evidence that the M318V change of the PK gene is associated with resistance to GCV and raise the question of cross resistance to this drug among different betaherpesviruses.

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