scholarly journals Thermodynamic Evidence for Negative Charge Stabilization by a Catalytic Metal Ion within an RNA Active Site

2011 ◽  
Vol 7 (2) ◽  
pp. 294-299 ◽  
Author(s):  
Raghuvir N. Sengupta ◽  
Daniel Herschlag ◽  
Joseph A. Piccirilli
Keyword(s):  
Active Site ◽  
Metal Ion ◽  
Negative Charge ◽  
Catalytic Metal ◽  
Charge Stabilization

scholarly journals Structural intermediates of a DNA–ligase complex illuminate the role of the catalytic metal ion and mechanism of phosphodiester bond formation

2019 ◽  
Vol 47 (14) ◽  
pp. 7147-7162 ◽  
Author(s):  
Adele Williamson ◽  
Hanna-Kirsti S Leiros
Keyword(s):  
Active Site ◽  
Metal Ion ◽  
Bond Formation ◽  
Strand Break ◽  
Nucleophilic Attack ◽  
Dna Ligase ◽  
Phosphodiester Bond ◽  
Dna Ligases ◽  
Catalytic Metal ◽  
Ternary Structure

Abstract DNA ligases join adjacent 5′ phosphate (5′P) and 3′ hydroxyl (3′OH) termini of double-stranded DNA via a three-step mechanism requiring a nucleotide cofactor and divalent metal ion. Although considerable structural detail is available for the first two steps, less is known about step 3 where the DNA-backbone is joined or about the cation role at this step. We have captured high-resolution structures of an adenosine triphosphate (ATP)-dependent DNA ligase from Prochlorococcus marinus including a Mn-bound pre-ternary ligase–DNA complex poised for phosphodiester bond formation, and a post-ternary intermediate retaining product DNA and partially occupied AMP in the active site. The pre-ternary structure unambiguously identifies the binding site of the catalytic metal ion and confirms both its role in activating the 3′OH terminus for nucleophilic attack on the 5′P group and stabilizing the pentavalent transition state. The post-ternary structure indicates that DNA distortion and most enzyme-AMP contacts remain after phosphodiester bond formation, implying loss of covalent linkage to the DNA drives release of AMP, rather than active site rearrangement. Additionally, comparisons of this cyanobacterial DNA ligase with homologs from bacteria and bacteriophage pose interesting questions about the structural origin of double-strand break joining activity and the evolution of these ATP-dependent DNA ligase enzymes.

Identification of an Active Site Ligand for a Group I Ribozyme Catalytic Metal Ion†

Biochemistry ◽  
2002 ◽  
Vol 41 (8) ◽  
pp. 2516-2525 ◽  
Author(s):  
Alexander A. Szewczak ◽  
Anne B. Kosek ◽  
Joseph A. Piccirilli ◽  
Scott A. Strobel
Keyword(s):  
Active Site ◽  
Metal Ion ◽  
Group I ◽  
Catalytic Metal

Structures of c-di-GMP/cGAMP degrading phosphodiesterase VcEAL: identification of a novel conformational switch and its implication

2019 ◽  
Vol 476 (21) ◽  
pp. 3333-3353 ◽  
Author(s):  
Malti Yadav ◽  
Kamalendu Pal ◽  
Udayaditya Sen
Keyword(s):  
Active Site ◽  
Metal Binding ◽  
Metal Ion ◽  
Triosephosphate Isomerase ◽  
Catalytic Mechanism ◽  
Degradation Mechanism ◽  
Structural Basis ◽  
Conserved Residues ◽  
Phosphodiester Bond ◽  
Cyclic Dinucleotides

Cyclic dinucleotides (CDNs) have emerged as the central molecules that aid bacteria to adapt and thrive in changing environmental conditions. Therefore, tight regulation of intracellular CDN concentration by counteracting the action of dinucleotide cyclases and phosphodiesterases (PDEs) is critical. Here, we demonstrate that a putative stand-alone EAL domain PDE from Vibrio cholerae (VcEAL) is capable to degrade both the second messenger c-di-GMP and hybrid 3′3′-cyclic GMP–AMP (cGAMP). To unveil their degradation mechanism, we have determined high-resolution crystal structures of VcEAL with Ca2+, c-di-GMP-Ca2+, 5′-pGpG-Ca2+ and cGAMP-Ca2+, the latter provides the first structural basis of cGAMP hydrolysis. Structural studies reveal a typical triosephosphate isomerase barrel-fold with substrate c-di-GMP/cGAMP bound in an extended conformation. Highly conserved residues specifically bind the guanine base of c-di-GMP/cGAMP in the G2 site while the semi-conserved nature of residues at the G1 site could act as a specificity determinant. Two metal ions, co-ordinated with six stubbornly conserved residues and two non-bridging scissile phosphate oxygens of c-di-GMP/cGAMP, activate a water molecule for an in-line attack on the phosphodiester bond, supporting two-metal ion-based catalytic mechanism. PDE activity and biofilm assays of several prudently designed mutants collectively demonstrate that VcEAL active site is charge and size optimized. Intriguingly, in VcEAL-5′-pGpG-Ca2+ structure, β5–α5 loop adopts a novel conformation that along with conserved E131 creates a new metal-binding site. This novel conformation along with several subtle changes in the active site designate VcEAL-5′-pGpG-Ca2+ structure quite different from other 5′-pGpG bound structures reported earlier.

scholarly journals HPF1 remodels the active site of PARP1 to enable the serine ADP-ribosylation of histones

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Fa-Hui Sun ◽  
Peng Zhao ◽  
Nan Zhang ◽  
Lu-Lu Kong ◽  
Catherine C. L. Wong ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Active Site ◽  
Negative Charge ◽  
Structural Data ◽  
Dna Breaks ◽  
Adp Ribosylation ◽  
Local Conformation ◽  
Key Residues ◽  
Structural Insights

AbstractUpon binding to DNA breaks, poly(ADP-ribose) polymerase 1 (PARP1) ADP-ribosylates itself and other factors to initiate DNA repair. Serine is the major residue for ADP-ribosylation upon DNA damage, which strictly depends on HPF1. Here, we report the crystal structures of human HPF1/PARP1-CAT ΔHD complex at 1.98 Å resolution, and mouse and human HPF1 at 1.71 Å and 1.57 Å resolution, respectively. Our structures and mutagenesis data confirm that the structural insights obtained in a recent HPF1/PARP2 study by Suskiewicz et al. apply to PARP1. Moreover, we quantitatively characterize the key residues necessary for HPF1/PARP1 binding. Our data show that through salt-bridging to Glu284/Asp286, Arg239 positions Glu284 to catalyze serine ADP-ribosylation, maintains the local conformation of HPF1 to limit PARP1 automodification, and facilitates HPF1/PARP1 binding by neutralizing the negative charge of Glu284. These findings, along with the high-resolution structural data, may facilitate drug discovery targeting PARP1.

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.

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