A Glycosyltransferase Inhibitor from a Molecular Fragment Library Simultaneously Interferes with Metal Ion and Substrate Binding

2012 ◽  
Vol 51 (17) ◽  
pp. 4171-4175 ◽  
Author(s):  
Rene Jørgensen ◽  
Lena Lisbeth Grimm ◽  
Nora Sindhuwinata ◽  
Thomas Peters ◽  
Monica M. Palcic
Keyword(s):  
Metal Ion ◽  
Substrate Binding ◽  
Molecular Fragment ◽  
Fragment Library

A Glycosyltransferase Inhibitor from a Molecular Fragment Library Simultaneously Interferes with Metal Ion and Substrate Binding

2012 ◽  
Vol 124 (17) ◽  
pp. 4247-4251 ◽  
Author(s):  
Rene Jørgensen ◽  
Lena Lisbeth Grimm ◽  
Nora Sindhuwinata ◽  
Thomas Peters ◽  
Monica M. Palcic
Keyword(s):  
Metal Ion ◽  
Substrate Binding ◽  
Molecular Fragment ◽  
Fragment Library

scholarly journals Characterization of the two 5-aminolaevulinic acid binding sites, the A- and P-sites, of 5-aminolaevulinic acid dehydratase from Escherichia coli

1995 ◽  
Vol 305 (1) ◽  
pp. 151-158 ◽  
Author(s):  
P Spencer ◽  
P M Jordan
Keyword(s):  
Schiff Base ◽  
Binding Sites ◽  
Metal Ion ◽  
Substrate Binding ◽  
Aminolaevulinic Acid ◽  
Acid Dehydratase ◽  
Carboxylate Groups ◽  
Catalytic Metal ◽  
A Site ◽  
Aminolaevulinic Acid Dehydratase

Experiments are described in which the individual properties of the two 5-aminolaevulinic acid (ALA) binding sites, the A-site and the P-site, of 5-aminolaevulinic acid dehydratase (ALAD) have been investigated. The ALA binding affinity at the A-site is greatly enhanced (at least 10-fold) on the binding of the catalytic metal ion (bound at the alpha-site). The nature of the catalytic metal ion, Mg2+ or Zn2+, also gave major variations in the substrate Km, P-site affinity for ALA, the effect of potassium and phosphate ions and the pH-dependence of substrate binding. Modification of the P-site by reaction of the enzyme-substrate Schiff base with NaBH4 and analysis of the reduced adduct by electro-spray mass spectrometry indicated a maximum of 1 mol of substrate incorporated/mol of subunit, correlating with a linear loss of enzyme activity. The reduced Schiff-base adduct was used to investigate substrate binding at the A-site by using rate-of-dialysis analysis. The affinity for ALA at the A-site of Mg alpha Zn beta ALAD was found to determine the Km for the reaction and was pH-dependent, with its affinity increasing from 1 mM at pH 6 to 70 microM at pH 8.5. The affinity of ALA at the P-site of Zn alpha An beta ALAD is proposed to limit the Km at pH values above 7, since the measured Kd for ALA at the A-site in 45 microM Tris, pH 8, was well below the observed Km (600 microM) under the same conditions. The amino group of the ALA molecule bound at the P-site was identified as a critical binding component for the A-site, explaining why ALA binding to ALAD is ordered, with the P-site ALA binding first. Structural requirements for ALA binding at the A- and P-sites have been identified: the P-site requires the carbonyl and carboxylate groups, whereas the A-site requires the amino, carbonyl and carboxylate groups of the substrate.

Metal ion and substrate binding to bovine galactosyltransferase

Biochemistry ◽  
1980 ◽  
Vol 19 (5) ◽  
pp. 929-934 ◽  
Author(s):  
Piet Jan Andree ◽  
Lawrence J. Berliner
Keyword(s):  
Metal Ion ◽  
Substrate Binding

scholarly journals Effect of Divalent Metal Ion on the Structure, Stability and Function of Klebsiella pneumoniae Nicotinate-Nucleotide Adenylyltransferase: Empirical and Computational Studies

2021 ◽  
Vol 23 (1) ◽  
pp. 116
Author(s):  
Olamide Jeje ◽  
Reabetswe Maake ◽  
Ruan van Deventer ◽  
Veruschka Esau ◽  
Emmanuel Amarachi Iwuchukwu ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Conformational Changes ◽  
Metal Ion ◽  
Therapeutic Potential ◽  
Divalent Cations ◽  
Homology Modelling ◽  
Substrate Binding ◽  
Structure Stability ◽  
Atp Binding ◽  
The Impact

The continuous threat of drug-resistant Klebsiella pneumoniae justifies identifying novel targets and developing effective antibacterial agents. A potential target is nicotinate nucleotide adenylyltransferase (NNAT), an indispensable enzyme in the biosynthesis of the cell-dependent metabolite, NAD+. NNAT catalyses the adenylation of nicotinamide/nicotinate mononucleotide (NMN/NaMN), using ATP to form nicotinamide/nicotinate adenine dinucleotide (NAD+/NaAD). In addition, it employs divalent cations for co-substrate binding and catalysis and has a preference for different divalent cations. Here, the biophysical structure of NNAT from K. pneumoniae (KpNNAT) and the impact of divalent cations on its activity, conformational stability and substrate-binding are described using experimental and computational approaches. The experimental study was executed using an enzyme-coupled assay, far-UV circular dichroism, extrinsic fluorescence spectroscopy, and thermal shift assays, alongside homology modelling, molecular docking, and molecular dynamic simulation. The structure of KpNNAT revealed a predominately α-helical secondary structure content and a binding site that is partially hydrophobic. Its substrates ATP and NMN share the same binding pocket with similar affinity and exhibit an energetically favourable binding. KpNNAT showed maximum activity and minimal conformational changes with Mg2+ as a cofactor compared to Zn2+, Cu2+ and Ni2+. Overall, ATP binding affects KpNNAT dynamics, and the dynamics of ATP binding depend on the presence and type of divalent cation. The data obtained from this study would serve as a basis for further evaluation towards designing structure-based inhibitors with therapeutic potential.

scholarly journals Mutational analysis of the zinc- and substrate-binding sites in the CphA metallo-β-lactamase from Aeromonas hydrophila

2008 ◽  
Vol 414 (1) ◽  
pp. 151-159 ◽  
Author(s):  
Carine Bebrone ◽  
Christine Anne ◽  
Frédéric Kerff ◽  
Gianpiero Garau ◽  
Kris De Vriendt ◽  
...  
Keyword(s):  
Significant Role ◽  
Metal Binding ◽  
Aeromonas Hydrophila ◽  
Metal Ion ◽  
Mutational Analysis ◽  
Substrate Binding ◽  
Zinc Ion ◽  
Negative Effect ◽  
Activity Spectrum ◽  
Substrate Binding Sites

The subclass B2 CphA (Carbapenemase hydrolysing Aeromonas) β-lactamase from Aeromonas hydrophila is a Zn2+-containing enzyme that specifically hydrolyses carbapenems. In an effort to evaluate residues potentially involved in metal binding and/or catalysis (His118, Asp120, His196 and His263) and in substrate specificity (Val67, Thr157, Lys224 and Lys226), site-directed mutants of CphA were generated and characterized. Our results confirm that the first zinc ion is in interaction with Asp120 and His263, and thus is located in the ‘cysteine’ zinc-binding site. His118 and His196 residues seem to be interacting with the second zinc ion, as their replacement by alanine residues has a negative effect on the affinity for this second metal ion. Val67 plays a significant role in the binding of biapenem and benzylpenicillin. The properties of a mutant with a five residue (LFKHV) insertion just after Val67 also reveals the importance of this region for substrate binding. This latter mutant has a higher affinity for the second zinc ion than wild-type CphA. The T157A mutant exhibits a significantly modified activity spectrum. Analysis of the K224Q and N116H/N220G/K224Q mutants suggests a significant role for Lys224 in the binding of substrate. Lys226 is not essential for the binding and hydrolysis of substrates. Thus the present paper helps to elucidate the position of the second zinc ion, which was controversial, and to identify residues important for substrate binding.

scholarly journals X-ray Crystallographic Studies of Substrate Binding to Aristolochene Synthase Suggest a Metal Ion Binding Sequence for Catalysis

2008 ◽  
Vol 283 (22) ◽  
pp. 15431-15439 ◽  
Author(s):  
Ekaterina Y. Shishova ◽  
Fanglei Yu ◽  
David J. Miller ◽  
Juan A. Faraldos ◽  
Yuxin Zhao ◽  
...  
Keyword(s):  
Metal Ion ◽  
Substrate Binding ◽  
Ion Binding ◽  
Metal Ion Binding ◽  
X Ray ◽  
Aristolochene Synthase ◽  
Binding Sequence

scholarly journals Structural characterization of tartrate dehydrogenase: a versatile enzyme catalyzing multiple reactions

2010 ◽  
Vol 66 (6) ◽  
pp. 673-684 ◽  
Author(s):  
Radhika Malik ◽  
Ronald E. Viola
Keyword(s):  
Active Site ◽  
Metal Ion ◽  
Substrate Binding ◽  
Hydride Transfer ◽  
Divalent Metal ◽  
Ammonium Ion ◽  
Hydroxyl Group ◽  
Metal Ion Binding ◽  
Cofactor Binding ◽  
Divalent Metal Ion

The first structure of an NAD-dependent tartrate dehydrogenase (TDH) has been solved to 2 Å resolution by single anomalous diffraction (SAD) phasing as a complex with the intermediate analog oxalate, Mg2+and NADH. This TDH structure fromPseudomonas putidahas a similar overall fold and domain organization to other structurally characterized members of the hydroxy-acid dehydrogenase family. However, there are considerable differences between TDH and these functionally related enzymes in the regions connecting the core secondary structure and in the relative positioning of important loops and helices. The active site in these complexes is highly ordered, allowing the identification of the substrate-binding and cofactor-binding groups and the ligands to the metal ions. Residues from the adjacent subunit are involved in both the substrate and divalent metal ion binding sites, establishing a dimer as the functional unit and providing structural support for an alternating-site reaction mechanism. The divalent metal ion plays a prominent role in substrate binding and orientation, together with several active-site arginines. Functional groups from both subunits form the cofactor-binding site and the ammonium ion aids in the orientation of the nicotinamide ring of the cofactor. A lysyl amino group (Lys192) is the base responsible for the water-mediated proton abstraction from the C2 hydroxyl group of the substrate that begins the catalytic reaction, followed by hydride transfer to NAD. A tyrosyl hydroxyl group (Tyr141) functions as a general acid to protonate the enolate intermediate. Each substrate undergoes the initial hydride transfer, but differences in substrate orientation are proposed to account for the different reactions catalyzed by TDH.

scholarly journals Unraveling the structural elements of pH sensitivity and substrate binding in the human zinc transporter SLC39A2 (ZIP2)

2019 ◽  
Vol 294 (20) ◽  
pp. 8046-8063 ◽  
Author(s):  
Gergely Gyimesi ◽  
Giuseppe Albano ◽  
Daniel G. Fuster ◽  
Matthias A. Hediger ◽  
Jonai Pujol-Giménez
Keyword(s):  
Binding Site ◽  
Metal Binding ◽  
In Silico ◽  
Metal Ion ◽  
Substrate Binding ◽  
Functional Characterization ◽  
Ph Sensitivity ◽  
Hek293 Cells ◽  
Substrate Binding Site

The transport and ion-coupling mechanisms of ZIP transporters remain largely uncharacterized. Previous work in our laboratory has revealed that the solute carrier family 39 member A2 (SLC39A2/ZIP2) increases its substrate transport rate in the presence of extracellular H+. Here, we used a combination of in silico and in vitro techniques involving structural modeling, mutagenesis, and functional characterization in HEK293 cells to identify amino acid residues potentially relevant for both the ZIP2–H+ interaction and substrate binding. Our ZIP2 models revealed a cluster of charged residues close to the substrate–translocation pore. Interestingly, the H63A substitution completely abrogated pH sensitivity, and substitutions of Glu-67 and Phe-269 altered the pH and voltage modulation of transport. In contrast, substitution of Glu-106, which might be part of a dimerization interface, altered pH but not voltage modulation. Substitution of Phe-269, located close to the substrate-binding site, also affected substrate selectivity. These findings were supported by an additional model of ZIP2 that was based on the structure of a prokaryotic homolog, Bordetella bronchiseptica ZrT/Irt-like protein (bbZIP), and in silico pKa calculations. We also found that residues Glu-179, His-175, His-202, and Glu-276 are directly involved in the coordination of the substrate metal ion. We noted that, unlike bbZIP, human ZIP2 is predicted to harbor a single divalent metal-binding site, with the charged side chain of Lys-203 replacing the second bound ion. Our results provide the first structural evidence for the previously observed pH and voltage modulation of ZIP2-mediated metal transport, identify the substrate-binding site, and suggest a structure-based transport mechanism for the ZIP2 transporter.

scholarly journals Wild-type and mutant d-xylose isomerase from Actinoplanes missouriensis: metal-ion dissociation constants, kinetic parameters of deuterated and non-deuterated substrates and solvent-isotope effects

1995 ◽  
Vol 307 (1) ◽  
pp. 135-142 ◽  
Author(s):  
P B M van Bastelaere ◽  
H L M Kersters-Hilderson ◽  
A M Lambeir
Keyword(s):  
Kinetic Parameters ◽  
Metal Ion ◽  
Dissociation Constants ◽  
Substrate Binding ◽  
Isotope Effects ◽  
Xylose Isomerase ◽  
Wild Type ◽  
Ion Dissociation ◽  
Solvent Isotope

The metal-ion dissociation constants (Mg2+, Mn2+) of wild-type and mutant D-xylose isomerases from Actinoplanes missouriensis have been determined by titrating the metal-ion-free enzymes with Mg2+ and Mn2+ respectively. Substitution of amino acids co-ordinated to metal-ion 1 (E181D, D245N) dramatically affects the dissociation constants, pH-activity profiles and apparent substrate binding. Mutagenesis of groups ligated to metal-ion 2 is less drastic except for that of Asp-255: a decrease in metal-ion affinity, a change in metal-ion preference and an improved apparent substrate binding (at pH values above the optimum), especially in the presence of Mn2+, are observed for the D255N enzyme. Similar effects, except for a slightly increased metal-ion affinity, are obtained by mutagenesis of the adjacent Glu-186 to Gln and the unconserved Ala-25 to Lys. Moreover, the striking acidic-pH shifts observed for the D255N and E186Q enzymes support the crucial role of the water molecule, Wa-690, Asp-255 and the adjacent Glu-186 in proton transfer from 2-OH to O-1 of the open and extended aldose substrate. Mutations of other important groups scarcely affect the metal-ion dissociation constants and pH-activity profiles, although pronounced effects on the kinetic parameters may be observed.

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