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

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.

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A second catalytic metal ion in a group I ribozyme

Nature ◽

10.1038/42076 ◽

1997 ◽

Vol 388 (6644) ◽

pp. 805-808 ◽

Cited By ~ 98

Author(s):

Lara B. Weinstein ◽

B. C. N. M. Jones ◽

Richard Cosstick ◽

Thomas R. Cech

Keyword(s):

Metal Ion ◽

Group I ◽

Catalytic Metal

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Functional Identification of Ligands for a Catalytic Metal Ion in Group I Introns†

Biochemistry ◽

10.1021/bi800519a ◽

2008 ◽

Vol 47 (26) ◽

pp. 6883-6894 ◽

Cited By ~ 23

Author(s):

Marcello Forconi ◽

Jihee Lee ◽

Jungjoon K. Lee ◽

Joseph A. Piccirilli ◽

Daniel Herschlag

Keyword(s):

Metal Ion ◽

Group I Introns ◽

Functional Identification ◽

Group I ◽

Catalytic Metal

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

ACS Chemical Biology ◽

10.1021/cb200202q ◽

2011 ◽

Vol 7 (2) ◽

pp. 294-299 ◽

Cited By ~ 3

Author(s):

Raghuvir N. Sengupta ◽

Daniel Herschlag ◽

Joseph A. Piccirilli

Keyword(s):

Active Site ◽

Metal Ion ◽

Negative Charge ◽

Catalytic Metal ◽

Charge Stabilization

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

Biochemical Journal ◽

10.1042/bcj20190399 ◽

2019 ◽

Vol 476 (21) ◽

pp. 3333-3353 ◽

Cited By ~ 3

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.

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