Functional analysis of amino acid residues at the dimerisation interface of KpnI DNA methyltransferase

2006 ◽  
Vol 387 (5) ◽  
pp. 515-523 ◽  
Author(s):  
Shivakumara Bheemanaik ◽  
Janusz M. Bujnicki ◽  
Valakunja Nagaraja ◽  
Desirazu N. Rao
Keyword(s):  
Dna Methyltransferase ◽  
Three Dimensional ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Three Dimensional Model ◽  
Cofactor Binding ◽  
Protein Fold Recognition ◽  
Fluorescence Measurements ◽  
Complete Disruption

AbstractKpnI DNA-(N6-adenine) methyltransferase (M.KpnI) recognises the sequence 5′-GGTACC-3′ and transfers the methyl group fromS-adenosyl-L-methionine (AdoMet) to the N6 position of the adenine residue in each strand. Earlier studies have shown that M.KpnI exists as a dimer in solution, unlike most other MTases. To address the importance of dimerisation for enzyme function, a three-dimensional model of M.KpnI was obtained based on protein fold-recognition analysis, using the crystal structures of M.RsrI and M.MboIIA as templates. Residues I146, I161 and Y167, the side chains of which are present in the putative dimerisation interface in the model, were targeted for site-directed mutagenesis. Methylation andin vitrorestriction assays showed that the mutant MTases are catalytically inactive. Mutation at the I146 position resulted in complete disruption of the dimer. The replacement of I146 led to drastically reduced DNA and cofactor binding. Substitution of I161 resulted in weakening of the interaction between monomers, leading to both monomeric and dimeric species. Steady-state fluorescence measurements showed that the wild-type KpnI MTase induces structural distortion in bound DNA, while the mutant MTases do not. The results establish that monomeric MTase is catalytically inactive and that dimerisation is an essential event for M.KpnI to catalyse the methyl transfer reaction.

scholarly journals A Three-dimensional Model of the Neprilysin 2 Active Site Based on the X-ray Structure of Neprilysin

2004 ◽  
Vol 279 (44) ◽  
pp. 46172-46181 ◽  
Author(s):  
Stéphanie Voisin ◽  
Didier Rognan ◽  
Claude Gros ◽  
Tanja Ouimet
Keyword(s):  
Active Site ◽  
Three Dimensional ◽  
Physiological Role ◽  
Site Directed Mutagenesis ◽  
Three Dimensional Model ◽  
X Ray ◽  
Amide Bonds ◽  
Inhibitory Compounds ◽  
Proposed Model

Neprilysin 2 (NEP2), a recently identified member of the M13 subfamily of metalloproteases, shares the highest degree of homology with the prototypical member of the family neprilysin. Whereas the study of thein vitroenzymatic activity of NEP2 shows that it resembles that of NEP as it cleaves the same substrates often at the same amide bonds and binds the same inhibitory compounds albeit with different potencies, its physiological role remains elusive because of the lack of selective inhibitors. To aid in the design of these novel compounds and better understand the different inhibitory patterns of NEP and NEP2, the x-ray structure of NEP was used as a template to build a model of the NEP2 active site. The results of our modeling suggest that the overall structure of NEP2 closely resembles that of NEP. The model of the active site reveals a 97% sequence identity with that of NEP with differences located within the S′2subsite of NEP2 where Ser133and Leu739replace two glycine residues in NEP. To validate the proposed model, site-directed mutagenesis was performed on a series of residues of NEP2, mutants expressed in AtT20 cells, and their ability to bind various substrates and inhibitory compounds was tested. The results confirm the involvement of the conserved Arg131and Asn567in substrate binding and catalytic activity of NEP2 and further show that the modifications in its S′2pocket, particularly the presence therein of Leu739, account for a number of differences in inhibitor binding between NEP and NEP2.

scholarly journals In Vivo and In Vitro Studies of Bacillus subtilis Ferrochelatase Mutants Suggest Substrate Channeling in the Heme Biosynthesis Pathway

2002 ◽  
Vol 184 (14) ◽  
pp. 4018-4024 ◽  
Author(s):  
Ulf Olsson ◽  
Annika Billberg ◽  
Sara Sjövall ◽  
Salam Al-Karadaghi ◽  
Mats Hansson
Keyword(s):  
Bacillus Subtilis ◽  
Amino Acid ◽  
Three Dimensional ◽  
Site Directed Mutagenesis ◽  
Dimensional Structure ◽  
Amino Acid Residues ◽  
Three Dimensional Structure ◽  
Activity Measurements

ABSTRACT Ferrochelatase (EC 4.99.1.1) catalyzes the last reaction in the heme biosynthetic pathway. The enzyme was studied in the bacterium Bacillus subtilis, for which the ferrochelatase three-dimensional structure is known. Two conserved amino acid residues, S54 and Q63, were changed to alanine by site-directed mutagenesis in order to detect any function they might have. The effects of these changes were studied in vivo and in vitro. S54 and Q63 are both located at helix α3. The functional group of S54 points out from the enzyme, while Q63 is located in the interior of the structure. None of these residues interact with any other amino acid residues in the ferrochelatase and their function is not understood from the three-dimensional structure. The exchange S54A, but not Q63A, reduced the growth rate of B. subtilis and resulted in the accumulation of coproporphyrin III in the growth medium. This was in contrast to the in vitro activity measurements with the purified enzymes. The ferrochelatase with the exchange S54A was as active as wild-type ferrochelatase, whereas the exchange Q63A caused a 16-fold reduction in V max. The function of Q63 remains unclear, but it is suggested that S54 is involved in substrate reception or delivery of the enzymatic product.

scholarly journals Cloning, post-translational modifications, heterologous expression and ligand-binding of boar salivary lipocalin

2000 ◽  
Vol 350 (2) ◽  
pp. 369-379 ◽  
Author(s):  
Dietrich LOEBEL ◽  
Andrea SCALONI ◽  
Sara PAOLINI ◽  
Carlo FINI ◽  
Lino FERRARA ◽  
...  
Keyword(s):  
Amino Acid ◽  
Sequence Similarity ◽  
Three Dimensional ◽  
Protein Isoforms ◽  
Cysteine Residues ◽  
Protein Chemistry ◽  
Single Individual ◽  
Amino Acid Residues ◽  
Three Dimensional Model ◽  
Submaxillary Glands

Boar submaxillary glands produce the sex-specific salivary lipocalin (SAL), which binds steroidal sex pheromones as endogenous ligands. The cDNA encoding SAL was cloned and sequenced. From a single individual, two protein isoforms, differing in three amino acid residues, were purified and structurally characterized by a combined Edman degradation/MS approach. These experiments ascertained that the mature polypeptide is composed of 168 amino acid residues, that one of the three putative glycosylation sites is post-translationally modified and the structure of the bound glycosidic moieties. Two of the cysteine residues are paired together in a disulphide bridge, whereas the remaining two occur as free thiols. SAL bears sequence similarity to other lipocalins; on this basis, a three-dimensional model of the protein has been built. A SAL isoform was expressed in Escherichiacoli in good yields. Protein chemistry and CD experiments verified that the recombinant product shows the same redox state at the cysteine residues and that the same conformation is observed as in the natural protein, thus suggesting similar folding. Binding experiments on natural and recombinant SAL were performed with the fluorescent probe 1-aminoanthracene, which was efficiently displaced by the steroidal sex pheromone, as well as by several odorants.

scholarly journals Functional Analysis of the Mycobacterium tuberculosis FAD-Dependent Thymidylate Synthase, ThyX, Reveals New Amino Acid Residues Contributing to an Extended ThyX Motif

2008 ◽  
Vol 190 (6) ◽  
pp. 2056-2064 ◽  
Author(s):  
Jonathan E. Ulmer ◽  
Yap Boum ◽  
Christopher D. Thouvenel ◽  
Hannu Myllykallio ◽  
Carol Hopkins Sibley
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Thymidylate Synthase ◽  
Three Dimensional ◽  
Pyrimidine Ring ◽  
Dimensional Structure ◽  
Directed Mutagenesis ◽  
Catalytic Residue ◽  
Amino Acid Residues ◽  
Insight Into

ABSTRACT A novel FAD-dependent thymidylate synthase, ThyX, is present in a variety of eubacteria and archaea, including the mycobacteria. A short motif found in all thyX genes, RHRX7-8S, has been identified. The three-dimensional structure of the Mycobacterium tuberculosis ThyX enzyme has been solved. Building upon this information, we used directed mutagenesis to produce 67 mutants of the M. tuberculosis thyX gene. Each enzyme was assayed to determine its ability to complement the defect in thymidine biosynthesis in a ΔthyA strain of Escherichia coli. Enzymes from selected strains were then tested in vitro for their ability to catalyze the oxidation of NADPH and the release of a proton from position 5 of the pyrimidine ring of dUMP. The results defined an extended motif of amino acids essential to enzyme activity in M. tuberculosis (Y44X24 H69X25R95HRX7 S105XRYX90R199 [with the underlined histidine acting as the catalytic residue and the underlined serine as the nucleophile]) and provided insight into the ThyX reaction mechanism. ThyX is found in a variety of bacterial pathogens but is absent in humans, which depend upon an unrelated thymidylate synthase, ThyA. Therefore, ThyX is a potential target for development of antibacterial drugs.

scholarly journals Mapping the Contact Sites of the Escherichia coli Division-Initiating Proteins FtsZ and ZapA by BAMG Cross-Linking and Site-Directed Mutagenesis

2018 ◽  
Vol 19 (10) ◽  
pp. 2928 ◽  
Author(s):  
Winfried Roseboom ◽  
Madhvi Nazir ◽  
Nils Meiresonne ◽  
Tamimount Mohammadi ◽  
Jolanda Verheul ◽  
...  
Keyword(s):  
Site Directed Mutagenesis ◽  
Cross Linking ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Binding Interface ◽  
Tetrameric Structure ◽  
Z Ring ◽  
Cross Links ◽  
Chemical Cross Linking

Cell division in bacteria is initiated by the polymerization of FtsZ at midcell in a ring-like structure called the Z-ring. ZapA and other proteins assist Z-ring formation and ZapA binds ZapB, which senses the presence of the nucleoids. The FtsZ–ZapA binding interface was analyzed by chemical cross-linking mass spectrometry (CXMS) under in vitro FtsZ-polymerizing conditions in the presence of GTP. Amino acids residue K42 from ZapA was cross-linked to amino acid residues K51 and K66 from FtsZ, close to the interphase between FtsZ molecules in protofilaments. Five different cross-links confirmed the tetrameric structure of ZapA. A number of FtsZ cross-links suggests that its C-terminal domain of 55 residues, thought to be largely disordered, has a limited freedom to move in space. Site-directed mutagenesis of ZapA reveals an interaction site in the globular head of the protein close to K42. Using the information on the cross-links and the mutants that lost the ability to interact with FtsZ, a model of the FtsZ protofilament–ZapA tetramer complex was obtained by information-driven docking with the HADDOCK2.2 webserver.

Inhibitory effect of resveratrol on the growth and angiogenesis of human endometrial tissue in an In Vitro three-dimensional model of endometriosis

2020 ◽  
Vol 20 (4) ◽  
pp. 484-490
Author(s):  
Mohammad Rasool Khazaei ◽  
Zahra Rashidi ◽  
Farzaneh Chobsaz ◽  
Elham Niromand ◽  
Mozafar Khazaei
Keyword(s):  
Three Dimensional ◽  
Inhibitory Effect ◽  
Endometrial Tissue ◽  
Dimensional Model ◽  
Three Dimensional Model

A three-dimensional model for the hammerhead ribozyme based on fluorescence measurements

Science ◽  
1994 ◽  
Vol 266 (5186) ◽  
pp. 785-789 ◽  
Author(s):  
T Tuschl ◽  
C Gohlke ◽  
T. Jovin ◽  
E Westhof ◽  
F Eckstein
Keyword(s):  
Hammerhead Ribozyme ◽  
Three Dimensional ◽  
Dimensional Model ◽  
Three Dimensional Model ◽  
Fluorescence Measurements

scholarly journals Apicomplexan co-infections impair with phagocytic activity in avian macrophages

2020 ◽  
Vol 119 (12) ◽  
pp. 4159-4168
Author(s):  
Runhui Zhang ◽  
Wanpeng Zheng ◽  
Arwid Daugschies ◽  
Berit Bangoura
Keyword(s):  
Laser Scanning ◽  
Three Dimensional ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Three Dimensional Model ◽  
Host Interactions ◽  
Macrophage Phagocytosis ◽  
High Doses ◽  
Free Ranging

AbstractMixed infections of Toxoplasma gondii and Eimeria tenella are likely to occur frequently due to the high prevalence of both pathogens in free-ranging chickens. In this study, we investigated the co-occurrence of the two parasites in the same immune-competent host cell towards altered patterns of parasite-host interactions. Chicken blood monocyte–derived macrophages were co-infected with T. gondii RH tachyzoites and E. tenella Houghton sporozoites in vitro for 24 h. Through monitoring the uptake of pH-sensitive pHrodo™ Zymosan BioParticles (“Zymosan”) by macrophages, we created a three-dimensional model and to analyze quantitatively phagocytosis using confocal laser scanning microscopy. Assessments of parasite populations were performed by qPCR at 2, 6, 12, and 24 h post-infection (hpi). At 6 hpi, phagocytosis was inhibited in the E. tenella–infected cultures while no inhibition of phagocytosis was observed due to T. gondii. Phagocytosis activity revealed more complex interactions during co-infection. At 12 and 24 hpi, phagocytosis response to “Zymosan” was distinctly weaker in co-infected cells than in all other groups except for cells mono-infected with high doses of E. tenella at 24 hpi. By qPCR, significantly reduced numbers of both intracellular parasites were recorded (10-fold) in all infected groups at 2 hpi. At 12 hpi, the T. gondii population reached lowest values but dramatically increased by 24 hpi. Our data confirm that macrophage phagocytosis is involved in the control of invasion by apicomplexan parasites in chicken which particularly applies to E. tenella infection and it was able to be altered by the co-existing parasites.

scholarly journals Identification of Novel Recognition Motifs and Regulatory Targets for the Yeast Actin-regulating Kinase Prk1p

2003 ◽  
Vol 14 (12) ◽  
pp. 4871-4884 ◽  
Author(s):  
Bo Huang ◽  
Guisheng Zeng ◽  
Alvin Y.J. Ng ◽  
Mingjie Cai
Keyword(s):  
Actin Cytoskeleton ◽  
Phosphorylation Site ◽  
Three Dimensional ◽  
Kinase Domain ◽  
Yeast Genome ◽  
Three Dimensional Model ◽  
Recognition Sequence ◽  
Yeast Saccharomyces Cerevisiae

Prk1p is a serine/threonine kinase involved in the regulation of the actin cytoskeleton organization in the yeast Saccharomyces cerevisiae. Previously, we have identified LxxQxTG as the phosphorylation site of Prk1p. In this report, the recognition sequence for Prk1p is investigated more thoroughly. It is found that the presence of a hydrophobic residue at the position of P-5 is necessary for Prk1p phosphorylation and L, I, V, and M are all able to confer the phosphorylation at various efficiencies. The residue flexibility at P-2 has also been identified to include Q, N, T, and S. A homology-based three-dimensional model of the kinase domain of Prk1p provided some structural interpretations for these substrate specificities. The characterization of the [L/I/V/M]xx[Q/N/T/S]xTG motif led to the identification of a spectrum of potential targets for Prk1p from yeast genome. One of them, Scd5p, which contains three LxxTxTG motifs and is previously known to be important for endocytosis and actin organization, has been chosen to demonstrate its relationship with Prk1p. Phosphorylation of Scd5p by Prk1p at the three LxxTxTG motifs could be detected in vitro and in vivo, and deletion of PRK1 suppressed the defects in actin cytoskeleton and endocytosis in one of the scd5 mutants. These results allowed us to conclude that Scd5p is likely another regulatory target of Prk1p.

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