scholarly journals Helicobacter pylori UreE, a urease accessory protein: specific Ni2+- and Zn2+-binding properties and interaction with its cognate UreG

2009 ◽  
Vol 422 (1) ◽  
pp. 91-100 ◽  
Author(s):  
Matteo Bellucci ◽  
Barbara Zambelli ◽  
Francesco Musiani ◽  
Paola Turano ◽  
Stefano Ciurli
Keyword(s):  
Helicobacter Pylori ◽  
Light Scattering ◽  
Metal Ion ◽  
Site Directed Mutagenesis ◽  
Size Exclusion ◽  
Titration Calorimetry ◽  
Directed Mutagenesis ◽  
Accessory Protein ◽  
Oligomeric State ◽  
Binding Properties

The persistence of Helicobacter pylori in the hostile environment of the human stomach is ensured by the activity of urease. The essentiality of Ni2+ for this enzyme demands proper intracellular trafficking of this metal ion. The metallo-chaperone UreE promotes Ni2+ insertion into the apo-enzyme in the last step of urease maturation while facilitating concomitant GTP hydrolysis. The present study focuses on the metal-binding properties of HpUreE (Helicobacter pylori UreE) and its interaction with the related accessory protein HpUreG, a GTPase involved in the assembly of the urease active site. ITC (isothermal titration calorimetry) showed that HpUreE binds one equivalent of Ni2+ (Kd=0.15 μM) or Zn2+ (Kd=0.49 μM) per dimer, without modification of the protein oligomeric state, as indicated by light scattering. Different ligand environments for Zn2+ and Ni2+, which involve crucial histidine residues, were revealed by site-directed mutagenesis, suggesting a mechanism for discriminating metal-ion-specific binding. The formation of a HpUreE–HpUreG protein complex was revealed by NMR spectroscopy, and the thermodynamics of this interaction were established using ITC. A role for Zn2+, and not for Ni2+, in the stabilization of this complex was demonstrated using size-exclusion chromatography, light scattering, and ITC experiments. A calculated viable structure for the complex suggested the presence of a novel binding site for Zn2+, actually detected using ITC and site-directed mutagenesis. The results are discussed in relation to available evidence of a UreE–UreG functional interaction in vivo. A possible role for Zn2+ in the Ni2+-dependent urease system is envisaged.

scholarly journals Biochemical and Phylogenetic Characterization of a Novel NADP+-Specific Isocitrate Dehydrogenase From the Marine Microalga Phaeodactylum tricornutum

2021 ◽  
Vol 8 ◽  
Author(s):  
Shiping Huang ◽  
Jiaxin Zhao ◽  
Wenjing Li ◽  
Peng Wang ◽  
Zhenglian Xue ◽  
...  
Keyword(s):  
Isocitrate Dehydrogenase ◽  
Metal Ion ◽  
Phaeodactylum Tricornutum ◽  
Site Directed Mutagenesis ◽  
Size Exclusion ◽  
Directed Mutagenesis ◽  
Transfer Event ◽  
Coenzyme Specificity ◽  
Type Iii ◽  
Marine Microalga

Isocitrate dehydrogenase (IDH) family of proteins is classified into three subfamilies, namely, types I, II, and III. Although IDHs are widely distributed in bacteria, archaea, and eukaryotes, all type III IDHs reported to date are found only in prokaryotes. Herein, a novel type III IDH subfamily member from the marine microalga Phaeodactylum tricornutum (PtIDH2) was overexpressed, purified, and characterized in detail for the first time. Relatively few eukaryotic genomes encode this type of IDH and PtIDH2 shares the highest homology with marine bacterial monomeric IDHs, suggesting that PtIDH2 originated through a horizontal gene transfer event between a marine alga and a bacterium. Size-exclusion chromatography revealed that the native PtIDH2 is a homotetramer (∼320 kDa) in solution, comprising four monomeric IDH-like subunits (80 kDa each). Enzymatic characterization showed that PtIDH2 is a bivalent metal ion-dependent enzyme and Mn2+ is the optimal activator. The recombinant PtIDH2 protein exhibited maximal activity at 35°C and pH 8.0 in the presence of Mn2+. Heat-inactivation analysis revealed that PtIDH2 is a cold-adapted enzyme. Kinetic analysis demonstrated that PtIDH2 is a completely NADP+-specific IDH with no detectable NAD+-associated catalytic activity. The three putative key NADP+-binding residues (His604, Arg615, and Arg664) in PtIDH2 were also evaluated by site-directed mutagenesis. The H604L/R615D/R664S triple mutant showed a 3.25-fold preference for NAD+ over NADP+, implying that the coenzyme specificity of PtIDH2 can be converted from NADP+ to NAD+ through rational engineering approaches. Additionally, the roles of the conserved residues Ala718 and Leu742 in the thermostability of PtIDH2 were also explored by site-directed mutagenesis. We found that the L742F mutant displayed higher thermostability than wild-type PtIDH2. This study expands the phylogeny of the IDH family and provides new insights into the evolution of IDHs.

scholarly journals The mechanism of action of dd-peptidases: the role of Threonine-299 and -301 in the Streptomyces R61 dd-peptidase

1994 ◽  
Vol 301 (2) ◽  
pp. 477-483 ◽  
Author(s):  
J M Wilkin ◽  
A Dubus ◽  
B Joris ◽  
J M Frère
Keyword(s):  
Amino Acids ◽  
Active Site ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Peptide Substrate ◽  
Binding Properties ◽  
Beta Lactamases ◽  
Penicillin Binding Proteins ◽  
Active Site Cavity

The side chains of residues Thr299 and Thr301 in the Streptomyces R61 DD-peptidase have been modified by site-directed mutagenesis. These amino acids are part of a beta-strand which forms a wall of the active-site cavity. Thr299 corresponds to the second residue of the Lys-Thr(Ser)-Gly triad, highly conserved in active-site beta-lactamases and penicillin-binding proteins (PBPs). Modification of Thr301 resulted only in minor alterations of the catalytic and penicillin-binding properties of the enzyme. No selective decrease of the rate of acylation was observed for any particular class of compounds. By contrast, the loss of the hydroxy group of the residue in position 299 yielded a seriously impaired enzyme. The rates of inactivation by penicillins were decreased 30-50-fold, whereas the reactions with cephalosporins were even more affected. The efficiency of hydrolysis against the peptide substrate was also seriously decreased. More surprisingly, the mutant was completely unable to catalyse transpeptidation reactions. The conservation of an hydroxylated residue in this position in PBPs is thus easily explained by these results.

scholarly journals Characterization of the Hepatitis C Virus NS2/3 Processing Reaction by Using a Purified Precursor Protein

2001 ◽  
Vol 75 (20) ◽  
pp. 9939-9946 ◽  
Author(s):  
Michele Pallaoro ◽  
Armin Lahm ◽  
Gabriella Biasiol ◽  
Mirko Brunetti ◽  
Caterina Nardella ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Cleavage Site ◽  
Metal Ion ◽  
Precursor Protein ◽  
Site Directed Mutagenesis ◽  
Size Exclusion ◽  
Sequence Alignments ◽  
Processing Rate

ABSTRACT The NS2-NS3 region of the hepatitis C virus polyprotein encodes a proteolytic activity that is required for processing of the NS2/3 junction. Membrane association of NS2 and the autocatalytic nature of the NS2/3 processing event have so far constituted hurdles to the detailed investigation of this reaction. We now report the first biochemical characterization of the self-processing activity of a purified NS2/3 precursor. Using multiple sequence alignments, we were able to define a minimal domain, devoid of membrane-anchoring sequences, which was still capable of performing the processing reaction. This truncated protein was efficiently expressed and processed in Escherichia coli. The processing reaction could be significantly suppressed by growth in minimal medium in the absence of added zinc ions, leading to the accumulation of an unprocessed precursor protein in inclusion bodies. This protein was purified to homogeneity, refolded, and shown to undergo processing at the authentic NS2/NS3 cleavage site with rates comparable to those observed using an in vitro-translated full-length NS2/3 precursor. Size-exclusion chromatography and a dependence of the processing rate on the concentration of truncated NS2/3 suggested a functional multimerization of the precursor protein. However, we were unable to observe trans cleavage activity between cleavage-site mutants and active-site mutants. Furthermore, the cleavage reaction of the wild-type protein was not inhibited by addition of a mutant that was unable to undergo self-processing. Site-directed mutagenesis data and the independence of the processing rate from the nature of the added metal ion argue in favor of NS2/3 being a cysteine protease having Cys993 and His952 as a catalytic dyad. We conclude that a purified protein can efficiently reproduce processing at the NS2/3 site in the absence of additional cofactors.

scholarly journals Transferrin-Binding Protein B of Neisseria meningitidis: Sequence-Based Identification of the Transferrin-Binding Site Confirmed by Site-Directed Mutagenesis

2004 ◽  
Vol 186 (3) ◽  
pp. 850-857 ◽  
Author(s):  
Geneviève Renauld-Mongénie ◽  
Laurence Lins ◽  
Tino Krell ◽  
Laure Laffly ◽  
Michèle Mignon ◽  
...  
Keyword(s):  
Neisseria Meningitidis ◽  
Binding Protein ◽  
Prediction Method ◽  
Binding Activity ◽  
Site Directed Mutagenesis ◽  
Titration Calorimetry ◽  
Directed Mutagenesis ◽  
Binding Domains ◽  
Homologous Strain ◽  
Protein B

ABSTRACT A sequence-based prediction method was employed to identify three ligand-binding domains in transferrin-binding protein B (TbpB) of Neisseria meningitidis strain B16B6. Site-directed mutagenesis of residues located in these domains has led to the identification of two domains, amino acids 53 to 57 and 240 to 245, which are involved in binding to human transferrin (htf). These two domains are conserved in an alignment of different TbpB sequences from N. meningitidis and Neisseria gonorrhoeae, indicating a general functional role of the domains. Western blot analysis and BIAcore and isothermal titration calorimetry experiments demonstrated that site-directed mutations in both binding domains led to a decrease or abolition of htf binding. Analysis of mutated proteins by circular dichroism did not provide any evidence for structural alterations due to the amino acid replacements. The TbpB mutant R243N was devoid of any htf-binding activity, and antibodies elicited by the mutant showed strong bactericidal activity against the homologous strain, as well as against several heterologous tbpB isotype I strains.

scholarly journals A Comparative Study on Nickel Binding to Hpn-like Polypeptides from Two Helicobacter pylori Strains

2021 ◽  
Vol 22 (24) ◽  
pp. 13210
Author(s):  
Danuta Witkowska ◽  
Agnieszka Szebesczyk ◽  
Joanna Wątły ◽  
Michał Braczkowski ◽  
Magdalena Rowińska-Żyrek
Keyword(s):  
Helicobacter Pylori ◽  
Comparative Study ◽  
Potentiometric Titration ◽  
Isothermal Titration Calorimetry ◽  
Titration Calorimetry ◽  
Binding Properties ◽  
Protein Fragments ◽  
Nickel Binding ◽  
Independent Reaction

Combined potentiometric titration and isothermal titration calorimetry (ITC) methods were used to study the interactions of nickel(II) ions with the N-terminal fragments and histidine-rich fragments of Hpn-like protein from two Helicobacter pylori strains (11637 and 26695). The ITC measurements were performed at various temperatures and buffers in order to extract proton-independent reaction enthalpies of nickel binding to each of the studied protein fragments. We bring up the problem of ITC results of nickel binding to the Hpn-like protein being not always compatible with those from potentiometry and MS regarding the stoichiometry and affinity. The roles of the ATCUN motif and multiple His and Gln residues in Ni(II) binding are discussed. The results provided the possibility to compare the Ni(II) binding properties between N-terminal and histidine-rich part of Hpn-like protein and between N-terminal parts of two Hpn-like strains, which differ mainly in the number of glutamine residues.

scholarly journals The human gut symbiont Ruminococcus gnavus shows specificity to blood group A antigen during mucin glycan foraging: Implication for niche colonisation in the gastrointestinal tract

PLoS Biology ◽  
2021 ◽  
Vol 19 (12) ◽  
pp. e3001498
Author(s):  
Haiyang Wu ◽  
Emmanuelle H. Crost ◽  
C David Owen ◽  
Wouter van Bakel ◽  
Ana Martínez Gascueña ◽  
...  
Keyword(s):  
Blood Group ◽  
Sequence Similarity ◽  
Glycoside Hydrolases ◽  
Site Directed Mutagenesis ◽  
Titration Calorimetry ◽  
Directed Mutagenesis ◽  
Human Gut ◽  
Blood Group A ◽  
Group A

The human gut symbiont Ruminococcus gnavus displays strain-specific repertoires of glycoside hydrolases (GHs) contributing to its spatial location in the gut. Sequence similarity network analysis identified strain-specific differences in blood-group endo-β-1,4-galactosidase belonging to the GH98 family. We determined the substrate and linkage specificities of GH98 from R. gnavus ATCC 29149, RgGH98, against a range of defined oligosaccharides and glycoconjugates including mucin. We showed by HPAEC-PAD and LC-FD-MS/MS that RgGH98 is specific for blood group A tetrasaccharide type II (BgA II). Isothermal titration calorimetry (ITC) and saturation transfer difference (STD) NMR confirmed RgGH98 affinity for blood group A over blood group B and H antigens. The molecular basis of RgGH98 strict specificity was further investigated using a combination of glycan microarrays, site-directed mutagenesis, and X-ray crystallography. The crystal structures of RgGH98 in complex with BgA trisaccharide (BgAtri) and of RgGH98 E411A with BgA II revealed a dedicated hydrogen network of residues, which were shown by site-directed mutagenesis to be critical to the recognition of the BgA epitope. We demonstrated experimentally that RgGH98 is part of an operon of 10 genes that is overexpresssed in vitro when R. gnavus ATCC 29149 is grown on mucin as sole carbon source as shown by RNAseq analysis and RT-qPCR confirmed RgGH98 expression on BgA II growth. Using MALDI-ToF MS, we showed that RgGH98 releases BgAtri from mucin and that pretreatment of mucin with RgGH98 confered R. gnavus E1 the ability to grow, by enabling the E1 strain to metabolise BgAtri and access the underlying mucin glycan chain. These data further support that the GH repertoire of R. gnavus strains enable them to colonise different nutritional niches in the human gut and has potential applications in diagnostic and therapeutics against infection.

scholarly journals Identification and characterization of the conserved nucleoside-binding sites in the Epstein-Barr virus thymidine kinase

2004 ◽  
Vol 379 (3) ◽  
pp. 795-803 ◽  
Author(s):  
Chung-Chun WU ◽  
Min-Che CHEN ◽  
Ya-Ru CHANG ◽  
Tsuey-Ying HSU ◽  
Jen-Yang CHEN
Keyword(s):  
Amino Acids ◽  
Binding Site ◽  
Thymidine Kinase ◽  
Crucial Role ◽  
Metal Ion ◽  
Epstein Barr Virus ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Barr Virus ◽  
Epstein Barr

Thymidine kinase (TK), encoded by EBV (Epstein–Barr virus), is an attractive target for antiviral therapy and provides a novel approach to the treatment of EBV-associated malignancies. Despite the extensive use of nucleoside analogues for the treatment of viral infections and cancer, the structure–function relationship of EBV TK has been addressed rarely. In the absence of any structural information, we sought to identify and elucidate the functional roles of amino acids in the nucleoside-binding site using site-directed mutagenesis. Through alignment with other human herpesviral TK protein sequences, we predicted that certain conserved regions comprise the nucleoside-binding site of EBV TK and, through site-directed mutagenesis, showed significant changes in activity and binding affinity for thymidine of site 3 (-DRH-) and 4 (-VFP-) mutants. For site 3, only mutants D392E (Asp392→Glu) and R393H retain activity, indicating that a negative charge is important for Asp392 and a positive charge is required for Arg393. The increased binding affinities of these two mutants for 3´-deoxy-2´,3´-didehydrothymidine suggest that the two residues are also important for substrate selection. Interestingly, the changed metal-ion usage pattern of D392E reveals that Asp392 plays multiple roles in this region. His394 cannot be compensated by other amino acids, also indicating a crucial role. In site 4, the F402Y mutant retains full activity; however, F402S retains only 60% relative activity. Strikingly, when Phe402 is substituted with serine residue, the original preferred pyrimidine substrates, such as 3´-azido-3´-deoxythymidine, iododeoxyuridine and β-l-5-iododioxolane uracil (l-form substrate), have decreased competitiveness with thymidine, suggesting that Phe402 plays a crucial role in substrate specificity and that the aromatic ring is important for function.

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