146-91-8Relevant academic research and scientific papers
Mechanistic Insights into the Metal-Dependent Activation of ZnII-Dependent Metallochaperones
Jordan, Matthew R.,Wang, Jiefei,Weiss, Andy,Skaar, Eric P.,Capdevila, Daiana A.,Giedroc, David P.
, p. 13661 - 13672 (2019)
Members of the COG0523 subfamily of candidate GTPase metallochaperones function in bacterial transition-metal homeostasis, but the nature of the cognate metal, mechanism of metal transfer, and identification of target protein(s) for metal delivery remain open questions. Here, we explore the multifunctionality of members of the subfamily linked to delivering ZnII to apoprotein targets under conditions of host-imposed transition-metal depletion. We examine two zinc-uptake repressor (Zur)-regulated COG0523 family members, each from a major human pathogen, Acinetobacter baumannii (AbZigA) and Staphylococcus aureus (SaZigA), in an effort to develop a model for ZnII metallochaperone activity. ZnII chelator competition experiments reveal one high-affinity (KZn1 ≈ 1010-1011 M-1) metal-binding site in each GTPase, while AbZigA and SaZigA are characterized by an additional one and two (lower-affinity) metal-binding sites, respectively. CoII titrations reveal that both metallochaperones have similar electronic absorption characteristics that indicate the presence of two tetrahedral metal coordination sites. High-affinity metal binding at the CXCC motif activates the GTPase activity of both enzymes, with ZnII more effective than CoII. Both GTPases bind the product, GDP, more tightly in the apoprotein than the ZnII-bound state and exhibit what is best described as a "locked" conformation around the GTP substrate. Negative thermodynamic linkage is observed between nucleotide binding and metal binding, leading to a new mechanistic model for COG0523-catalyzed metal delivery.
What is the conformation of physiologically-active dinucleoside polyphosphates in solution? Conformational analysis of free dinucleoside polyphosphates by NMR and molecular dynamics simulations
Stern, Noa,Major, Dan Thomas,Gottlieb, Hugo Emilio,Weizman, Daniel,Fischer, Bilha
, p. 4637 - 4652 (2010)
Dinucleoside polyphosphates, or dinucleotides (NpnN′; N, N′ = A, U, G, C; n = 2-7), are naturally occurring ubiquitous physiologically active compounds. Despite the interest in dinucleotides, and the relevance of their conformation to their biological function, the conformation of dinucleotides has been insufficiently studied. Therefore, here we performed conformational analysis of a series of NpnN′ Na+ salts (N = A, G, U, C; N′ = A, G, U, C; n = 2-5) by various NMR techniques. All studied dinucleotides, except for Up4/5U, formed intramolecular base stacking interactions in aqueous solutions as indicated by NMR. The conformation around the glycosidic angle in NpnN′s was found to be anti/high anti and the preferred conformation around the C4′-C5′, C5′-O5′ bonds was found to be gauche-gauche (gg). The ribose moiety in NpnN′s showed a small preference for the S conformation, but when attached to cytosine the ribose ring preferred the N conformation. However, no predominant conformation was observed for the ribose moiety in any of the dinucleotides. Molecular dynamics simulations of Ap2A and Ap4A Na+ salts supported the experimental results. In addition, three modes of base-stacking were found for Ap2/4A: α-α, β-β and α-β, which exist in equilibrium, while none is dominant. We conclude that natural, free NpnN′s (n = 2-5) at physiological pH exist mostly in a folded (stacked), rather than extended conformation, in several interconverting stacking modes. Intramolecular base stacking of NpnN′s does not alter the conformation of each of the nucleotide moieties, which remains the same as that of the mononucleotides in solution.
One-Pot Synthesis of α,γ-Dinucleoside 5'-Triphosphates, G5'pppG and A5'pppA, Using S,S'-Bis(4-chlorophenyl)phosphorodithioate
Fukuoka, Koichiro,Suda, Fuminori,Suzuki, Ryo,Ishikawa, Masahide,Hata, Tsujiaki
, p. 499 - 502 (1994)
S,S'-Bis(4-chlorophenyl) phosphorodithioate was useful for the synthesis of α,γ-dinucleoside 5'-triphosphates, G5'pppG and A5'pppA starting from the corresponding unprotected nucleoside 5'-phosphates under neutral conditions. G5'pppG was used for the synthesis of m7G5'pppG by means of the N7-methylation of one of two guanine moieties of G5'pppG.
Catalytic activity of human guanylate-binding protein 1 coupled to the release of structural restraints imposed by the C-terminal domain
Ince, Semra,Zhang, Ping,Kutsch, Miriam,Krenczyk, Oktavian,Shydlovskyi, Sergii,Herrmann, Christian
, p. 582 - 599 (2021)
Human guanylate-binding protein 1 (hGBP-1) shows a dimer-induced acceleration of the GTPase activity yielding GDP as well as GMP. While the head-to-head dimerization of the large GTPase (LG) domain is well understood, the role of the rest of the protein, particularly of the GTPase effector domain (GED), in dimerization and GTP hydrolysis is still obscure. In this study, with truncations and point mutations on hGBP-1 and by means of biochemical and biophysical methods, we demonstrate that the intramolecular communication between the LG domain and the GED (LG:GED) is crucial for protein dimerization and dimer-stimulated GTP hydrolysis. In the course of GTP binding and γ-phosphate cleavage, conformational changes within hGBP-1 are controlled by a chain of amino acids ranging from the region near the nucleotide-binding pocket to the distant LG:GED interface and lead to the release of the GED from the LG domain. This opening of the structure allows the protein to form GED:GED contacts within the dimer, in addition to the established LG:LG interface. After releasing the cleaved γ-phosphate, the dimer either dissociates yielding GDP as the final product or it stays dimeric to further cleave the β-phosphate yielding GMP. The second phosphate cleavage step, that is, the formation of GMP, is even more strongly coupled to structural changes and thus more sensitive to structural restraints imposed by the GED. Altogether, we depict a comprehensive mechanism of GTP hydrolysis catalyzed by hGBP-1, which provides a detailed molecular understanding of the enzymatic activity connected to large structural rearrangements of the protein. Database: Structural data are available in RCSB Protein Data Bank under the accession numbers: 1F5N, 1DG3, 2B92.
Donor substrate binding and enzymatic mechanism of human core α1,6-fucosyltransferase (FUT8)
K?tzler, Miriam P.,Blank, Simon,Bantleon, Frank I.,Spillner, Edzard,Meyer, Bernd
, p. 1915 - 1925 (2012)
Background: Fucosylation is essential for various biological processes including tumorigenesis, inflammation, cell-cell recognition and host-pathogen interactions. Biosynthesis of fucosylated glycans is accomplished by fucosyltransferases. The enzymatic product of core α1,6-fucosyltransferase (FUT8) plays a major role in a plethora of pathological conditions, e.g. in prognosis of hepatocellular carcinoma and in colon cancer. Detailed knowledge of the binding mode of its substrates is required for the design of molecules that can modulate the activity of the enzyme. Methods: We provide a detailed description of binding interactions of human FUT8 with its natural donor substrate GDP-fucose and related compounds. GDP-Fuc was placed in FUT8 by structural analogy to the structure of protein-O-fucosyltransferase (cePOFUT) co-crystallized with GDP-Fuc. The epitope of the donor substrate bound to FUT8 was determined by STD NMR. The in silico model is further supported by experimental data from SPR binding assays. The complex was optimized by molecular dynamics simulations. Results: Guanine is specifically recognized by His363 and Asp453. Furthermore, the pyrophosphate is tightly bound via numerous hydrogen bonds and contributes affinity to a major part. Arg365 was found to bind both the β-phosphate and the fucose moiety at the same time. Conclusions: Discovery of a novel structural analogy between cePOFUT and FUT8 allows the placement of the donor substrate GDP-Fuc. The positioning was confirmed by various experimental and computational techniques. General significance: The model illustrates details of the molecular basis of substrate recognition for a human fucosyltransferase for the first time and, thus, provides a basis for structure-based design of inhibitors.
The glycosyltransferase involved in thurandacin biosynthesis catalyzes both O- and S-glycosylation
Wang, Huan,Oman, Trent J.,Zhang, Ran,Garcia De Gonzalo, Chantal V.,Zhang, Qi,Van Der Donk, Wilfred A.
, p. 84 - 87 (2014)
The S-glycosyltransferase SunS is a recently discovered enzyme that selectively catalyzes the conjugation of carbohydrates to the cysteine thiol of proteins. This study reports the discovery of a second S-glycosyltransferase, ThuS, and shows that ThuS catalyzes both S-glycosylation of the thiol of cysteine and O-glycosylation of the hydroxyl group of serine in peptide substrates. ThuS-catalyzed S-glycosylation is more efficient than O-glycosylation, and the enzyme demonstrates high tolerance with respect to both nucleotide sugars and peptide substrates. The biosynthesis of the putative products of the thuS gene cluster was reconstituted in vitro, and the resulting S-glycosylated peptides thurandacin A and B exhibit highly selective antimicrobial activity toward Bacillus thuringiensis.
THE PREPARATION OF ADENOSINE 5'-PYROPHOSPHATE BY A NON-ENZYMIC METHOD.
DAWSON,FORD,EICHBERG
, p. 104 - 106 (1965)
1. A non-enzymic method for the preparation of adenosine 5'-diphosphate
Phosphorylation of ganciclovir phosphonate by cellular GMP kinase determines the stereoselectivity of anti-human cytomegalovirus activity
Miller, Wayne H.,Beauchamp, Lilia M.,Meade, Eric,Reardon, John E.,Biron, Karen K.,Smith, Albert A.,Goss, Charles A.,Miller, Richard L.
, p. 341 - 356 (2000)
A racemic mixture of ganciclovir phosphonate was resolved by stereoselective phosphorylation using GMP kinase. The R-enantiomer of ganciclovir phosphonate was active against human cytomegalovirus but the S- enantiomer was less active. We show that enantiomeric selectivity of antiviral activity for ganciclovir phosphonate was conferred by stereoselective phosphorylations by mammalian enzymes, not by stereoslective inhibition of DNA polymerase from human cytomegalovirus.
Functionally nonequivalent interactions of guanosine 5'-triphosphate, inosine 5'-triphosphate, and xanthosine 5'-triphosphate with the retinal G-protein, transducin, and with G(i)-proteins in HL-60 leukemia cell membranes
Klinker, Jan F.,Seifert, Roland
, p. 551 - 562 (1997)
G proteins mediate signal transfer from receptors to effector systems. In their guanosine 5'-triphosphate (GTP) bound form, G-protein α-subunits activate effector systems. Termination of G-protein activation is achieved by the high-affinity GTPase [E.C. 3.6.1.-] of their α-subunits. Like GTP, inosine 5' -triphosphate (ITP) and xanthosine 5' triphosphate (XTP) can support effector system activation. We studied the interactions of GTP, ITP, and XTP with the retinal G protein, transducin (TD), and with G-proteins in HL-60 leukemia cell membranes. TD hydrolyzed nucleoside 5'-triphosphates (NTPs) in the order of efficacy GTP > ITP > XTP. NTPs eluted TD from rod outer segment disk membranes in the same order of efficacy. ITP and XTP competitively inhibited TD catalyzed GTP hydrolysis. In HL-60 membranes, the chemoattractants N-formyl-L-methionyl-L-leucyl-L- phenylaline (fMLP) and leukotriene B4 (LTB4,) effectively activated GTP and ITP hydrolysis by G(i) proteins. fMLP and LTB4, were at least l0-fold more potent activators of ITPase than of GTPasc. Complement C5a effectively activated the GTPase of G(i)-proteins but was only a weak stimulator of ITPase. The potency of C5a to activate GTP and ITP hydrolysis was similar. The fMLP stimulated GTPase had a lower K(m) value than the fMLP-stimulated ITPase, whereas the opposite was true for the V(max) values. fMLP, C5a, and LTB4 did not stimulate XTP hydrolysis. Collectively, our data show that GTP, ITP, and XTP bind to G-proteins with different affinities, that G-proteins hydrolyze NTPs with different efficacies, and that chemoattractants stimulate GTP and ITP hydrolysis by G(i)-proteins in a receptor-specific manner. On the basis of our results and the data in the literature, we put forward the hypothesis that GTP, ITP, and XTP act as differential signal amplifiers and signal sorters at the G-protein level.
Biochemical properties of the human guanylate binding protein 5 and a tumor-specific truncated splice variant
Wehner, Mark,Herrmann, Christian
, p. 1597 - 1605 (2010)
The human guanylate binding protein 5 (hGBP5) belongs to the family of interferon-γ-inducible large GTPases, which are well known for their high induction by pro-inflammatory cytokines. The cellular role of this protein family is unclear at this point, but there are indications for antiviral and antibacterial activity of hGBP1. hGBP5 exists in three splice variants, forming two different proteins, of which the tumor-specific one is C-terminally truncated by 97 amino acids, and therefore lacks the CaaX motif for geranylgeranylation. Here we present biochemical data on the splice variants of hGBP5. We show that, unlike hGBP1, hGBP5a/b and hGBP5ta do not bind GMP or produce any GMP during hydrolysis despite the fact the residues involved in GMP production from hGBP1 are conserved in hGBP5. Hydrolysis of GTP is concentration-dependent and shows weak self-activation. Thermodynamic studies showed strongly negative entropic changes during nucleotide binding, which reflect structural ordering in the protein during nucleotide binding. These structural changes were also observed during changes in the oligomerization state. We observed only a minor influence of the C-terminal truncation on hydrolysis, nucleotide binding and oligomerization of hGBP5. Based on these similarities we speculate that the missing C-terminal part, which also carries the geranylgeranylation motif, is the reason for the dysregulation of hGBP5′s function in lymphoma cells.
