How to complete the tautomerization and substrate-assisted activation prior to C–C bond fission by meta-cleavage product hydrolase LigY?

2020 ◽  
Vol 10 (17) ◽  
pp. 5856-5869
Author(s):  
Junjie Wang ◽  
Xiaowen Tang ◽  
Yixin Zhang ◽  
Yanwei Li ◽  
Ledong Zhu ◽  
...  
Keyword(s):  
Cleavage Product ◽  
The Other ◽  
Binding Modes ◽  
Bidentate Mode

Two feasible binding modes could complete the C–C bond fission of the substrate. One is the bidentate mode and five-coordination, and the other is the monodentate mode and five-coordination.

scholarly journals Accurate processing of human pre-rRNA in vitro.

1989 ◽  
Vol 9 (10) ◽  
pp. 4422-4431 ◽  
Author(s):  
G J Hannon ◽  
P A Maroney ◽  
A Branch ◽  
B J Benenfield ◽  
H D Robertson ◽  
...  
Keyword(s):  
Hela Cell ◽  
18S Rrna ◽  
Cleavage Product ◽  
The Other ◽  
Second Step ◽  
Cleavage Sites ◽  
Initial Cleavage ◽  
External Transcribed Spacer ◽  
Cyclic Phosphate

We report here that the mature 5' terminus of human 18S rRNA is generated in vitro by a two-step processing reaction. In the first step, SP6 transcripts were specifically cleaved in HeLa cell nucleolar extract at three positions near the external transcribed spacer (ETS)-18S boundary. Of these cleavage sites, two were major and the other was minor. RNase T1 fingerprint and secondary nuclease analyses placed the two major cleavage sites 3 and 8 bases upstream from the mature 5' end of 18S rRNA and the minor cleavage site 1 base into the 18S sequence. All three cleavages yielded 5'-hydroxyl, 2'-3'-cyclic phosphate termini and were 5' of adenosine residues in the sequence UACCU, which was repeated three times near the ETS-18S boundary. In the second step, the initial cleavage product containing 3 bases of ETS was converted to an RNA with a 5' terminus identical to that of mature 18S RNA by an activity found in HeLa cell cytoplasmic extracts.

scholarly journals Structural and Biochemical Studies on the Regulation of Biotin Carboxylase by Substrate Inhibition and Dimerization

2011 ◽  
Vol 286 (27) ◽  
pp. 24417-24425 ◽  
Author(s):  
Chi-Yuan Chou ◽  
Liang Tong
Keyword(s):  
Binding Sites ◽  
Analytical Ultracentrifugation ◽  
Substrate Inhibition ◽  
Kinetic Studies ◽  
The Other ◽  
Biotin Carboxylase ◽  
Binding Modes ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Dimer Interface

Biotin carboxylase (BC) activity is shared among biotin-dependent carboxylases and catalyzes the Mg-ATP-dependent carboxylation of biotin using bicarbonate as the CO2 donor. BC has been studied extensively over the years by structural, kinetic, and mutagenesis analyses. Here we report three new crystal structures of Escherichia coli BC at up to 1.9 Å resolution, complexed with different ligands. Two structures are wild-type BC in complex with two ADP molecules and two Ca2+ ions or two ADP molecules and one Mg2+ ion. One ADP molecule is in the position normally taken by the ATP substrate, whereas the other ADP molecule occupies the binding sites of bicarbonate and biotin. One Ca2+ ion and the Mg2+ ion are associated with the ADP molecule in the active site, and the other Ca2+ ion is coordinated by Glu-87, Glu-288, and Asn-290. Our kinetic studies confirm that ATP shows substrate inhibition and that this inhibition is competitive against bicarbonate. The third structure is on the R16E mutant in complex with bicarbonate and Mg-ADP. Arg-16 is located near the dimer interface. The R16E mutant has only a 2-fold loss in catalytic activity compared with the wild-type enzyme. Analytical ultracentrifugation experiments showed that the mutation significantly destabilized the dimer, although the presence of substrates can induce dimer formation. The binding modes of bicarbonate and Mg-ADP are essentially the same as those to the wild-type enzyme. However, the mutation greatly disrupted the dimer interface and caused a large re-organization of the dimer. The structures of these new complexes have implications for the catalysis by BC.

A three-dimensional zinc-based coordination polymer built from 5-carboxylato-1-carboxylatomethyl-2-oxidopyridinium with a 4-connectedsratopology

2014 ◽  
Vol 70 (8) ◽  
pp. 801-804
Author(s):  
De-Yun Ma ◽  
Jing Zheng ◽  
Jie-Qiong Cao ◽  
Xu-Min Lin ◽  
You-Biao Ling
Keyword(s):  
Coordination Polymer ◽  
Luminescence Spectrum ◽  
Room Temperature ◽  
Three Dimensional ◽  
The Other ◽  
Planar Structure ◽  
Zigzag Chain ◽  
Carboxylate Group ◽  
Aromatic Carboxylate ◽  
Bidentate Mode

A novel three-dimensional ZnIIcomplex, poly[aqua(μ4-5-carboxylato-1-carboxylatomethyl-2-oxidopyridinium)zinc(II)], [Zn(C8H5NO4)(H2O)]n, has been prepared by hydrothermal assembly of Zn(CH3COO)2·2H2O and 5-carboxy-1-(carboxymethyl)pyridin-1-ium-2-olate (H2ccop). The ccop2−anions bridge the ZnIIcations in a head-to-tail fashionviamonodentate aromatic carboxylate and phenolate O atoms to form an extended zigzag chain which runs parallel to the [011] direction. One O atom of the aliphatic carboxylate group of the ccop2−ligand coordinates to the ZnIIatom of a neighbouring chain thereby producing undulating layers which lie parallel to the (01-1) plane. A similar parallel undulating planar structure can be obtained if a path involving the other O atom of the aliphatic carboxylate group is considered. Thus, the aliphatic carboxylate group acts in a bridging bidentate mode to give extended –Zn–O–C–O–Zn– sequences running parallel to [001] which link the layers into an overall three-dimensional framework. The three-dimensional framework can be simplified as a 4-connected sra topology with a Schläfli symbol of 42.63.8 if all the ZnIIcentres and ccop2−anions are regarded as tetrahedral 4-connected nodes. The three-dimensional luminescence spectrum was measured at room temperature with excitation and emission wavelengths of 344–354 and 360–630 nm, respectively, at intervals of 0.15 and 2 nm, respectively.

Accurate processing of human pre-rRNA in vitro

1989 ◽  
Vol 9 (10) ◽  
pp. 4422-4431
Author(s):  
G J Hannon ◽  
P A Maroney ◽  
A Branch ◽  
B J Benenfield ◽  
H D Robertson ◽  
...  
Keyword(s):  
Hela Cell ◽  
18S Rrna ◽  
Cleavage Product ◽  
The Other ◽  
Second Step ◽  
Cleavage Sites ◽  
Initial Cleavage ◽  
External Transcribed Spacer ◽  
Cyclic Phosphate

We report here that the mature 5' terminus of human 18S rRNA is generated in vitro by a two-step processing reaction. In the first step, SP6 transcripts were specifically cleaved in HeLa cell nucleolar extract at three positions near the external transcribed spacer (ETS)-18S boundary. Of these cleavage sites, two were major and the other was minor. RNase T1 fingerprint and secondary nuclease analyses placed the two major cleavage sites 3 and 8 bases upstream from the mature 5' end of 18S rRNA and the minor cleavage site 1 base into the 18S sequence. All three cleavages yielded 5'-hydroxyl, 2'-3'-cyclic phosphate termini and were 5' of adenosine residues in the sequence UACCU, which was repeated three times near the ETS-18S boundary. In the second step, the initial cleavage product containing 3 bases of ETS was converted to an RNA with a 5' terminus identical to that of mature 18S RNA by an activity found in HeLa cell cytoplasmic extracts.

STUDY OF METHYLENE BLUE INTERACTION WITH SYNTHETIC POLYNUCLEOTIDE POLY(rA)-POLY(rU)

2020 ◽  
Vol 54 (2 (252)) ◽  
pp. 112-117
Author(s):  
M.A. Parsadanyan ◽  
M.A. Shahinyan ◽  
A.P. Antonyan
Keyword(s):  
Methylene Blue ◽  
Fluorescence Spectroscopy ◽  
The Other ◽  
Binding Modes ◽  
Other Hand ◽  
Non Linear ◽  
Order Of Magnitude ◽  
Synthetic Polynucleotide ◽  
Specific Ligand

A study of the interaction of methylene blue (MB) with poly(rA)-poly(rU) by the method of fluorescence spectroscopy has been carried out. The data obtained revealed that MB, being a DNA-specific ligand, can bind to double-stranded regions of RNA. In this regard, as in the case of DNA, semi-intercalation was the most preferable mechanism for the binding of this ligand to poly(rA)-poly(rU). On the other hand, non-linear curves of dependence of F0/F on concentration of polynucleotide might result from two binding modes, the second of which was probably of an electrostatic nature. Proceeding from the data obtained, the value of KSV was revealed to be almost an order of magnitude less than for DNA, which may indicate that RNA is a less preferable target for MB.

The αEC domain of human fibrinogen-420 is a stable and early plasmin cleavage product

Blood ◽  
2000 ◽  
Vol 95 (7) ◽  
pp. 2297-2303 ◽  
Author(s):  
Dianne Applegate ◽  
Lara Stoike Steben ◽  
Kathe M. Hertzberg ◽  
Gerd Grieninger
Keyword(s):  
Gel Electrophoresis ◽  
Degradation Products ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cleavage Product ◽  
The Other ◽  
Cross Linking ◽  
Human Fibrinogen ◽  
Western Blots ◽  
Sds Page

Abstract Human fibrinogen-420, (Eβγ)2, was isolated from plasma and evaluated for its ability to form clots and for its susceptibility to proteolysis. Clotting parameters, including cross-linking of subunit chains, of this subclass and of the more abundant fibrinogen-340 (βγ)2, were found to be similar, suggesting little impact of the unique EC domains of fibrinogen-420 on coagulation. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis of plasmic digestion patterns revealed production from fibrinogen-420 of the conventional fibrinogen degradation products, X, Y, D, and E, to be comparable to that from fibrinogen-340 in all respects except the presence of at least 2 additional cleavage products that were shown by Western blot analysis to contain the EC domain. One was a stable fragment (ECX) comigrating with a 34-kd yeast recombinant EC domain, and the other was an apparent precursor. Their release occurred early, before that of fragments D and E. Two bands of the same mobility and antibody reactivity were found in Western blots of plasma collected from patients with myocardial infarction shortly after the initiation of thrombolytic therapy.

scholarly journals BIOLOGICAL ACTIVITY OF THE CLEAVAGE PRODUCT OF HUMAN IMMUNOGLOBULIN G WITH CYANOGEN BROMIDE

1967 ◽  
Vol 125 (5) ◽  
pp. 787-805 ◽  
Author(s):  
Mira Lahav ◽  
Ruth Arnon ◽  
Michael Sela
Keyword(s):  
Biological Activity ◽  
Cyanogen Bromide ◽  
Biological Activities ◽  
Gel Filtration ◽  
Cleavage Product ◽  
The Other ◽  
Antibody Activity ◽  
Peptide Bonds ◽  
Binding Reaction ◽  
Therapeutic Uses

Treatment of human IgG with cyanogen bromide in 0.05 M HCl under specified conditions resulted in the cleavage of about half of its methionyl peptide bonds. A major fragment of about 5S was isolated from the reaction mixture by gel filtration in quantitative yield. The CNBr fragment reacted fully with goat antiserum against human light chain, but its reaction with anti-heavy chain was markedly decreased. The treatment with CNBr caused a drastic decrease in the following biological activities of IgG: complement fixing, skin binding, reaction with antiglobulin factors, and reaction with specific anti-Gm(12) serum. On the other hand, the reaction with serum of anti-Gm(1) or anti-Gm(4) specificity was not impaired and antibody activity, namely antistreptolysin and isohemagglutinin, was retained after the treatment with CNBr. It is concluded that the CNBr cleaves preferentially the methionyl bonds in the Fc portion of IgG, and that the major fragment obtained, denoted F(ab'')2, has still the combining properties of a divalent antibody. The possible therapeutic uses of F(ab'')2 are discussed.

DNA Binding Diversity Achieved Through the Interaction of GATA1 N-Finger and GATA Motif Is Important for Embryonic Erythropoiesis

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3441-3441
Author(s):  
Atsushi Hasegawa ◽  
Ritsuko Shimizu ◽  
Hirofumi Kurokawa ◽  
Masayuki Yamamoto
Keyword(s):  
Dna Binding ◽  
Target Genes ◽  
Conflicts Of Interest ◽  
Consensus Sequence ◽  
Late Gestation ◽  
The Other ◽  
Binding Modes ◽  
Substitution Mutations ◽  
Gata Motif

Abstract Abstract 3441 Transcription factor GATA1 regulates a set of genes essential for the erythroid and megakaryocytic cell differentiation through the interaction with GATA motifs (consensus sequence: A/TGATAA/T). Two zinc fingers within GATA1 have been identified to be important in the DNA binding of GATA1, which are referred to as C-finger (CF) and N-finger (NF) domains. It has been shown that transactivation activity of GATA1 is completely abolished upon deletion of the CF domain, indicating that the CF domain is a requisite for the DNA binding of GATA1. While conventional reporter transactivation analyses hardly clarified the importance of the NF domain for the DNA binding, substitution mutations on 216th arginine (R216) located in the DNA-interacting surface of the NF domain have been identified to cause familial diseases of thrombocytopenia, thalassemia, and porphyria. As a consequence of the substitution of R216 to glutamine (Q) or tryptophan (W), DNA binding activity of GATA1 to a palindromic configuration of two GATA motifs (palindromic GATA) was largely diminished, while that to a single GATA motif was maintained. In this study we have examined the DNA binding diversity of GATA1 caused by the difference in the configuration of GATA motifs. We performed surface plasmon resonance (SPR) analyses of GATA1 to a single GATA, a palindromic GATA, and a repeating configuration of two GATA motifs (tandem GATA). We found that GATA1 binds to the palindromic GATA motif in a bivalent way, while it binds to the single GATA motif in a monovalent mode. We also found that a double quantity of GATA1 is associated with the tandem GATA motif and GATA1 lacking the NF domain binds to any configurations of GATA motif in a monovalent way. To further investigate contribution of the NF domain to the binding mode of GATA1, we have constructed two types of GATA1 mutants; one type was the substitution mutations on R216 (R216Q and R216W) that were mouse homologues of the human mutations, while the other type was the alanine substitution mutation on three lysine residues (K245, K246 and K312; referred to as 3KA mutant), whereby dimerization potential of GATA1 was reduced to trace level similar to the case for GATA1 lacking the NF domain. Impotantly, R216Q and R216W mutants bind the palindromic GATA motif in a monovalent way, while these mutants bind normally to the other configuration of GATA motifs. In contrast, we found that one molecule of 3KA mutant bound to the tandem GATA motif and this observation seems to explain well the fact that dimerization potential of GATA1 is an important requisite for the full-function of GATA1 in embryos. The binding modes of this 3KA mutant to the other configurations were not influenced. These results thus demonstrate that the both NF and CF domains recognize the multiple configurations of GATA motifs and specify the binding modes of GATA1. Importantly, GATA1-deficient mice rescued with R216Q were lethal during late gestation period due to abnormality in erythroid differentiation, indicating that the contribution of the NF domain to the recognition of the palindromic GATA motif configuration indeed functions in vivo. These results thus support our contention that the NF domain acts to regulate a proper spatio-temporal gene expression of a subset of GATA1 target genes utilizing the variations in the GATA motif configuration. Disclosures: No relevant conflicts of interest to declare.

The αEC domain of human fibrinogen-420 is a stable and early plasmin cleavage product

Blood ◽  
2000 ◽  
Vol 95 (7) ◽  
pp. 2297-2303 ◽  
Author(s):  
Dianne Applegate ◽  
Lara Stoike Steben ◽  
Kathe M. Hertzberg ◽  
Gerd Grieninger
Keyword(s):  
Gel Electrophoresis ◽  
Degradation Products ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cleavage Product ◽  
The Other ◽  
Cross Linking ◽  
Human Fibrinogen ◽  
Western Blots ◽  
Sds Page

Human fibrinogen-420, (Eβγ)2, was isolated from plasma and evaluated for its ability to form clots and for its susceptibility to proteolysis. Clotting parameters, including cross-linking of subunit chains, of this subclass and of the more abundant fibrinogen-340 (βγ)2, were found to be similar, suggesting little impact of the unique EC domains of fibrinogen-420 on coagulation. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis of plasmic digestion patterns revealed production from fibrinogen-420 of the conventional fibrinogen degradation products, X, Y, D, and E, to be comparable to that from fibrinogen-340 in all respects except the presence of at least 2 additional cleavage products that were shown by Western blot analysis to contain the EC domain. One was a stable fragment (ECX) comigrating with a 34-kd yeast recombinant EC domain, and the other was an apparent precursor. Their release occurred early, before that of fragments D and E. Two bands of the same mobility and antibody reactivity were found in Western blots of plasma collected from patients with myocardial infarction shortly after the initiation of thrombolytic therapy.

Stromatoporoids from the Famennian (Devonian) Wabamun Formation, Normandville oilfield, north–central Alberta, Canada

1988 ◽  
Vol 62 (03) ◽  
pp. 411-419 ◽  
Author(s):  
Colin W. Stearn
Keyword(s):  
Patch Reef ◽  
Abundant Species ◽  
The Other ◽  
North Central ◽  
Biotic Crisis

Stromatoporoids are the principal framebuilding organisms in the patch reef that is part of the reservoir of the Normandville field. The reef is 10 m thick and 1.5 km2in area and demonstrates that stromatoporoids retained their ability to build reefal edifices into Famennian time despite the biotic crisis at the close of Frasnian time. The fauna is dominated by labechiids but includes three non-labechiid species. The most abundant species isStylostroma sinense(Dong) butLabechia palliseriStearn is also common. Both these species are highly variable and are described in terms of multiple phases that occur in a single skeleton. The other species described areClathrostromacf.C. jukkenseYavorsky,Gerronostromasp. (a columnar species), andStromatoporasp. The fauna belongs in Famennian/Strunian assemblage 2 as defined by Stearn et al. (1988).

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