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 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 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.

Molecular Cloning, Modeling, and Site-Directed Mutagenesis of Type III Polyketide Synthase from Sargassum binderi (Phaeophyta)

2010 ◽  
Vol 13 (5) ◽  
pp. 845-856 ◽  
Author(s):  
Hariyanti Baharum ◽  
Hiroyuki Morita ◽  
Akifumi Tomitsuka ◽  
Fong-Chin Lee ◽  
Kim-Yong Ng ◽  
...  
Keyword(s):  
Molecular Cloning ◽  
Polyketide Synthase ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Type Iii Polyketide Synthase ◽  
Type Iii ◽  
Sargassum Binderi

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.

scholarly journals Mapping of a copper-binding site on the small CP12 chloroplastic protein of Chlamydomonas reinhardtii using top-down mass spectrometry and site-directed mutagenesis

2009 ◽  
Vol 419 (1) ◽  
pp. 75-86 ◽  
Author(s):  
Jenny Erales ◽  
Brigitte Gontero ◽  
Julian Whitelegge ◽  
Frédéric Halgand
Keyword(s):  
Metal Ion ◽  
Calvin Cycle ◽  
Computational Modelling ◽  
Copper Ion ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Copper Binding ◽  
Metal Ion Binding ◽  
Top Down ◽  
Copper Ligands

CP12 is a small chloroplastic protein involved in the Calvin cycle that was shown to bind copper, a metal ion that is involved in the transition of CP12 from a reduced to an oxidized state. In order to describe CP12's copper-binding properties, copper-IMAC experiments and site-directed mutagenesis based on computational modelling, were coupled with top-down MS [electrospray-ionization MS and MS/MS (tandem MS)]. Immobilized-copper-ion-affinity-chromatographic experiments allowed the primary characterization of the effects of mutation on copper binding. Top-down MS/MS experiments carried out under non-denaturing conditions on wild-type and mutant CP12–Cu2+ complexes then allowed fragment ions specifically binding the copper ion to be determined. Comparison of MS/MS datasets defined three regions involved in metal ion binding: residues Asp16–Asp23, Asp38–Lys50 and Asp70–Glu76, with the two first regions containing selected residues for mutation. These data confirmed that copper ligands involved glutamic acid and aspartic residues, a situation that contrasts with that obtaining for typical protein copper chelators. We propose that copper might play a role in the regulation of the biological activity of CP12.

scholarly journals A type III CRISPR ancillary ribonuclease degrades its cyclic oligoadenylate activator

2019 ◽  
Author(s):  
Januka S. Athukoralage ◽  
Shirley Graham ◽  
Sabine Grüschow ◽  
Christophe Rouillon ◽  
Malcolm F. White
Keyword(s):  
Viral Infection ◽  
Active Site ◽  
Thermus Thermophilus ◽  
Growth Arrest ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Type Iii ◽  
Secondary Messengers ◽  
Infected Cells ◽  
Higher Eukaryotes

Cyclic oligoadenylate (cOA) secondary messengers are generated by type III CRISPR systems in response to viral infection. cOA allosterically activates the CRISPR ancillary ribonucleases Csx1/Csm6, which degrade RNA non-specifically using a HEPN (Higher Eukaryotes and Prokaryotes, Nucleotide binding) active site. This provides effective immunity, but can also lead to growth arrest in infected cells, necessitating a means to deactivate the ribonuclease once viral infection has been cleared. In the crenarchaea, dedicated ring nucleases degrade cA4(cOA consisting of 4 AMP units), but the equivalent enzyme has not been identified in bacteria. We demonstrate that, inThermus thermophilusHB8, the uncharacterised protein TTHB144 is a cA4-activated HEPN ribonuclease that also degrades its activator. TTHB144 binds and degrades cA4at an N-terminal CARF (CRISPR Associated Rossman Fold) domain. The two activities can be separated by site-directed mutagenesis. TTHB144 is thus the first example of a self-limiting CRISPR ribonuclease.Graphical abstract

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