A Reappraisal, Based on <sup>31</sup>P NMR, of the Direct Coordination of a Metal Ion with the Phosphoryl Oxygen at the Cleavage Site of a Hammerhead Ribozyme

Suzumura, Ken Ichi; Yoshinari, Koichi; Tanaka, Yoshiyuki; Takagi, Yoko; Kasai, Yusuke; Warashina, Masaki; Kuwabara, Tomoko; Orita, Masaya; Taira, Kazunari

doi:10.1021/ja0202098

Cited by 22 publications

(31 citation statements)

References 44 publications

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“…For the same P9 substitution but in a background of 10 mM Mg 2+ , Peracchi et al (1997) found an 8.8-fold increased K 1/2 for stimulating Cd 2+ . Metal coordination to the A9/G10.1 site in activity-inhibited constructs has also been spectroscopically measured, providing further support for the description of the A9 interaction as a ground-state interaction (Horton et al 1998;Hunsicker and DeRose 2000;Suzumura et al 2002;Vogt et al 2006). Additionally, in the present study, the K 1/2 values for Cd 2+ activity stimulation in the WT and S p constructs likely represent low estimates of the actual K 1/ 2 values in this ionic background.…”

Section: Resultssupporting

confidence: 70%

Ground-state coordination of a catalytic metal to the scissile phosphate of a tertiary-stabilized Hammerhead ribozyme

Ward¹,

DeRose²

2011

RNA

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Although the Hammerhead ribozyme (HHRz) has long been used as a model system in the field of ribozyme enzymology, several details of its mechanism are still not well understood. In particular, significant questions remain concerning the disposition and role of catalytic metals in the HHRz. Previous metal-rescue experiments using a ''minimal'' HHRz resulted in prediction of a catalytic metal that is bound in the A9/G10.1 site in the ground state of the reaction and that bridges to the scissile phosphate further along the reaction pathway. ''Native'' or extended HHRz constructs contain tertiary contacts that stabilize a more compact structure at moderate ionic strength. We performed Cd 2+ rescue experiments on an extended HHRz from Schistosoma mansoni using stereo-pure scissile phosphorothioate-substituted substrates in order to determine whether a metal ion makes contact with the scissile phosphate in the ground state or further along the reaction coordinate. Inhibition in Ca 2+ /Mg 2+ and rescue by thiophilic Cd 2+ was specific for the R p -S stereoisomer of the scissile phosphate. The affinity of the rescuing Cd 2+ , measured in two different ionic strength backgrounds, increased fourfold to 17-fold when the pro-R p oxygen is replaced by sulfur. These data support a model in which the rescuing metal ion makes a ground-state interaction with the scissile phosphate in the native HHRz. The resulting model for Mg 2+ activation in the HHRz places a metal ion in contact with the scissile phosphate, where it may provide ground-state electrostatic activation of the substrate.

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Section: Resultssupporting

confidence: 70%

Ground-state coordination of a catalytic metal to the scissile phosphate of a tertiary-stabilized Hammerhead ribozyme

Ward¹,

DeRose²

2011

RNA

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“…Also here, phosphorothioate studies helped to prove the coordination of divalent metal ions during catalysis [271,272]. resonance, respectively [274][275][276][277]. The catalytic activity of the hammerhead ribozyme was also shown to be in principle independent of divalent metal ions [278] 113 Cd NMR, which allow to observe both the RNA liganding sites and the metal ion simultaneously.…”

Section: Cadmium(ii)-rescue Experimentsmentioning

confidence: 87%

Complex Formation of Cadmium with Sugar Residues, Nucleobases, Phosphates, Nucleotides, and Nucleic Acids

Sigel

Skilandat

Sigel

et al. 2012

Metal Ions in Life Sciences

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Cadmium(II), commonly classified as a relatively soft metal ion, prefers indeed aromaticnitrogen sites (e.g., N7 of purines) over oxygen sites (like sugar-hydroxyl groups). However, matters are not that simple, though it is true that the affinity of Cd(2+) towards ribose-hydroxyl groups is very small; yet, a correct orientation brought about by a suitable primary binding site and a reduced solvent polarity, as it is expected to occur in a folded nucleic acid, may facilitate metal ion-hydroxyl group binding very effectively. Cd(2+) prefers the guanine(N7) over the adenine(N7), mainly because of the steric hindrance of the (C6)NH(2) group in the adenine residue. This Cd(2+)-(N7) interaction in a guanine moiety leads to a significant acidification of the (N1)H meaning that the deprotonation reaction occurs now in the physiological pH range. N3 of the cytosine residue, together with the neighboring (C2)O, is also a remarkable Cd(2+) binding site, though replacement of (C2)O by (C2)S enhances the affinity towards Cd(2+) dramatically, giving in addition rise to the deprotonation of the (C4)NH(2) group. The phosphodiester bridge is only a weak binding site but the affinity increases further from the mono-to the di-and the triphosphate. The same also holds for the corresponding nucleotides. Complex stability of the pyrimidine-nucleotides is solely determined by the coordination tendency of the phosphate group(s), whereas in the case of purine-nucleotides macrochelate formation takes place by the interaction of the phosphate-coordinated Cd(2+) with N7. The extents of the formation degrees of these chelates are summarized and the effect of a non-bridging sulfur atom in a thiophosphate group (versus a normal phosphate group) is considered. Mixed ligand complexes containing a nucleotide and a further mono-or bidentate ligand are covered and it is concluded that in these species N7 is released from the coordination sphere of Cd(2+). In the case that the other ligand contains an aromatic residue (e.g., 2,2'-bipyridine or the indole ring of tryptophanate) intramolecular stack formation takes place. With buffers like Tris or Bistris mixed ligand complexes are formed. Cd(2+) coordination to dinucleotides and to dinucleoside monophosphates provides some insights regarding the interaction between Cd(2+) and nucleic acids. Cd(2+) binding to oligonucleotides follows the principles of coordination to its units. The available crystal studies reveal that N7 of purines is the prominent binding site followed by phosphate oxygens and other heteroatoms in nucleic acids. Due to its high thiophilicity, Cd(2+) is regularly used in so-called thiorescue experiments, which lead to the identification of a direct involvement of divalent metal ions in ribozyme catalysis.

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“…According to the HSAB (Hard and Soft, Acid and Base) rule [21], a reduction in the cleavage rate of a 'soft' thio-substituted substrate should be observed in the presence of 'hard' Mg 2+ cation (the thio effect), and restoration of a normal cleavage rate of a sulfur-containing substrate should occur in the presence of thiophilic cations such as Mn , in increasing order (the rescue effect). Analysis of these types of interaction led to a better understanding of the mechanistic aspects of the action of naturally occurring catalytic ribozymes: group I and II introns [22][23][24][25], the RNA subunit of RNase P [26,27], and the hammerhead ribozymes [18,[28][29][30]. The successful application of Pchiral phosphorothioates in those mechanistic studies prompted us to establish the role of phosphate groups in the catalytic core of deoxyribozyme [10][11][12][13][14][15][16][17][18][19][20][21][22][23].…”

Section: +mentioning

confidence: 99%

Mapping of the functional phosphate groups in the catalytic core of deoxyribozyme 10–23

et al. 2007

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The RNA-cleaving DNA enzymes, like most ribozymes, require a divalent metal cation for their cleavage activity [1]. Among metal ion-dependent DNA enzymes, deoxyribozyme 10-23, first selected and characterized by Santoro & Joyce [1,2], has been examined most extensively both in vitro and in vivo [3][4][5]. This enzyme consists of a 15-nucleotide conserved catalytic core and variable substrate recognition arms (Fig. 1A). Cleavage of an RNA substrate is highly sequence-specific, and occurs between the bulged 5¢-purine and paired 3¢-pyrimidine nucleosides, resulting in the formation of the two products, a 5¢-terminal product with a 2¢,3¢-cyclic phosphate, and a 3¢-terminal product containing an OH group at its 5¢-end. The enzyme preferentially uses Mg 2+ for its activity, although other divalent metal ions are accepted as cofactors [1,2,6]. To date, the structure of the substrate-deoxyribozyme 10-23 active complex remains unknown [7,8], and the mechanistic details of the catalytic reaction are not fully understood. Therefore, much effort has been devoted to determine the role of individual nucleotides in the 10-23 catalytic core, as well as their relative importance [9-13]. Despite numerous studies performed on a mutant deoxyribozyme 10-23 containing chemical modifications inserted into the catalytic core, the role of particular phosphates within this domain has not been investigated in detail. We have studied this issue by systematic modification of each phosphate of the core with phosphorothioate (PS) analogs, in which one of the two nonbridging oxygen atoms of the phosphate group was replaced with a sulfur atom. The RNA phosphodiester bond cleavage activity of a series of 16 thio-deoxyribozymes 10-23, containing a P-stereorandom single phosphorothioate linkage in predetermined positions of the catalytic core from P1 to P16, was evaluated under single-turnover conditions in the presence of either 3 mm Mg 2+ or 3 mm Mn 2+. A metal-specificity switch approach permitted the identification of nonbridging phosphate oxygens (proR P or proS P ) located at seven positions of the core (P2, P4 and P9-13) involved in direct coordination with a divalent metal ion(s). By contrast, phosphorothioates at positions P3, P6, P7 and P14-16 displayed no functional relevance in the deoxyribozyme-mediated catalysis. Interestingly, phosphorothioate modifications at positions P1 or P8 enhanced the catalytic efficiency of the enzyme. Among the tested deoxyribozymes, thio-substitution at position P5 had the largest deleterious effect on the catalytic rate in the presence of Mg 2+ , and this was reversed in the presence of Mn 2+. Further experiments with thio-deoxyribozymes of stereodefined P-chirality suggested direct involvement of both oxygens of the P5 phosphate and the proR P oxygen at P9 in the metal ion coordination. In addition, it was found that the oxygen atom at C6 of G 6 contributes to metal ion binding and that this interaction is essential for 10-23 deoxyribozyme catalytic activity.Abbreviations AP, 2-aminopurine; DNAzyme, RNA-...

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A Reappraisal, Based on ³¹P NMR, of the Direct Coordination of a Metal Ion with the Phosphoryl Oxygen at the Cleavage Site of a Hammerhead Ribozyme

Cited by 22 publications

References 44 publications

Ground-state coordination of a catalytic metal to the scissile phosphate of a tertiary-stabilized Hammerhead ribozyme

Ground-state coordination of a catalytic metal to the scissile phosphate of a tertiary-stabilized Hammerhead ribozyme

Complex Formation of Cadmium with Sugar Residues, Nucleobases, Phosphates, Nucleotides, and Nucleic Acids

Mapping of the functional phosphate groups in the catalytic core of deoxyribozyme 10–23

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A Reappraisal, Based on 31P NMR, of the Direct Coordination of a Metal Ion with the Phosphoryl Oxygen at the Cleavage Site of a Hammerhead Ribozyme

Cited by 22 publications

References 44 publications

Ground-state coordination of a catalytic metal to the scissile phosphate of a tertiary-stabilized Hammerhead ribozyme

Ground-state coordination of a catalytic metal to the scissile phosphate of a tertiary-stabilized Hammerhead ribozyme

Complex Formation of Cadmium with Sugar Residues, Nucleobases, Phosphates, Nucleotides, and Nucleic Acids

Mapping of the functional phosphate groups in the catalytic core of deoxyribozyme 10–23

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A Reappraisal, Based on ³¹P NMR, of the Direct Coordination of a Metal Ion with the Phosphoryl Oxygen at the Cleavage Site of a Hammerhead Ribozyme