Understanding the Acid–Base Properties of Adenosine: The Intrinsic Basicities of N1, N3 and N7

Kapinos, Larisa E.; Operschall, Bert P.; Larsen, Erik; Sigel, Helmut

doi:10.1002/chem.201003544

Cited by 71 publications

(85 citation statements)

References 56 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We found that twister does not have a HB donor with good geometry and a chemical group with a reasonable pK A to act as a general acid. The only HB donor with reasonable geometry is the N3 of A10, but this nitrogen has a very low pK A of about -4.2 to 1.0 (22,23). Although this would be a great chemical group to interact with the 5′-OH leaving group, it would require a highly perturbed pK A to act as a general acid.…”

Section: Discussionmentioning

confidence: 99%

Structural basis for the fast self-cleavage reaction catalyzed by the twister ribozyme

Eiler¹,

Wang²,

Steitz

2014

Proc. Natl. Acad. Sci. U.S.A.

142

View full text Add to dashboard Cite

Twister is a recently discovered RNA motif that is estimated to have one of the fastest known catalytic rates of any naturally occurring small self-cleaving ribozyme. We determined the 4.1-Å resolution crystal structure of a twister sequence from an organism that has not been cultured in isolation, and it shows an ordered scissile phosphate and nucleotide 5′ to the cleavage site. A second crystal structure of twister from Orzyza sativa determined at 3.1-Å resolution exhibits a disordered scissile phosphate and nucleotide 5′ to the cleavage site. The core of twister is stabilized by base pairing, a large network of stacking interactions, and two pseudoknots. We observe three nucleotides that appear to mediate catalysis: a guanosine that we propose deprotonates the 2′-hydroxyl of the nucleotide 5′ to the cleavage site and a conserved adenosine. We suggest the adenosine neutralizes the negative charge on a nonbridging phosphate oxygen atom at the cleavage site. The active site also positions the labile linkage for in-line nucleophilic attack, and thus twister appears to simultaneously use three strategies proposed for small self-cleaving ribozymes. The twister crystal structures (i) show its global structure, (ii) demonstrate the significance of the double pseudoknot fold, (iii) provide a possible hypothesis for enhanced catalysis, and (iv) illuminate the roles of all 10 highly conserved nucleotides of twister that participate in the formation of its small and stable catalytic pocket.X-ray crystallography | RNA structure | weak derivative phasing | samarium derivatives | cesium derivatives T he twister RNA motif was identified by bioinformatic searches and then validated biochemically to be a small self-cleaving ribozyme (1). This recently discovered class of ribozymes is called twister because its conserved secondary structure resembles the ancient Egyptian hieroglyph "twisted flax." Representatives of the twister ribozyme class are found in all domains of life, but its biological role has yet to be determined. In addition to twister, the small self-cleaving ribozyme family includes the hammerhead, hairpin, hepatitis delta virus (HDV), Varkud satellite (VS), and glmS ribozymes (the glmS ribozyme is upsteam of the the glmS gene that codes for the enzyme that catalyzes glucosamine-6-phosphate production) (1-6).The small self-cleaving ribozyme family can be split into two groups based on whether their active site is formed by an irregular helix (hammerhead, hairpin, and VS) or a double pseudoknot (PK) structure (HDV and glmS) (7). The structures of HDV and glmS are known, whereas twister was predicted from representative sequences to use two PKs to form its active site. It was expected that twister would be smaller in size than either HDV or glmS and more comparable in size and complexity to hammerhead (1).The self-cleavage rate constant of twister is estimated to be as rapid as or slightly more rapid than the hammerhead ribozyme. The estimated rate constants (k obs ) for twister is 1,000 per minute, and the experime...

show abstract

Section: Discussionmentioning

confidence: 99%

Structural basis for the fast self-cleavage reaction catalyzed by the twister ribozyme

Eiler¹,

Wang²,

Steitz

2014

Proc. Natl. Acad. Sci. U.S.A.

142

View full text Add to dashboard Cite

show abstract

“…4.3 [31,35]), the (N1)H + sides 9 of adenosines (pK a ca. 3.8 [31,36]) as well as the (N7)H + of guanosines (pK a ca. 2.5 [31,37,38]),…”

Section: Insert Figure 2 Close To Here (Width: 6 Cm)mentioning

confidence: 99%

“…Note, the basicity, and thus the metal ion affinity, of N3 should not be underestimated: The micro acidity constant for the (N3)H + site in otherwise neutral adenosine was estimated as pk a/(N3)H = 1.5 ± 0.3 [36]; such a value is ideal for outersphere interactions [59][60][61], e.g., with Mg 2+ (see the discussed solid-state structure of Mg(2'AMP) in Section 3.2).…”

mentioning

confidence: 99%

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

Sigel

Skilandat

Sigel

et al. 2012

Metal Ions in Life Sciences

Self Cite

View full text Add to dashboard Cite

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.

show abstract

“…In general, protonation occurs preferentially at N1 of adenine, with minor populations of protonation at N7 and N3. [29] It is one of our future aims to verify the precise location of protonation using the corresponding 15 N-single atom labeled A6. [30,31] In this context, we note that G48 (G62 in O. sativa twister ribozyme) has been suggested to act as general base and to activate the attacking 2′-OH.…”

mentioning

confidence: 99%

A Mini‐Twister Variant and Impact of Residues/Cations on the Phosphodiester Cleavage of this Ribozyme Class

Košutić¹,

Neuner²,

Ren

et al. 2015

Angewandte Chemie

View full text Add to dashboard Cite

Nucleolytic ribozymes catalyze site-specific cleavage of their phosphodiester backbones. A minimal version of the twister ribozyme is reported that lacks the phylogenetically conserved stem P1 while retaining wild-type activity. Atomic mutagenesis revealed that nitrogen atoms N1 and N3 of the adenine-6 at the cleavage site are indispensable for cleavage. By NMR spectroscopy, a pK a value of 5.1 was determined for a 13 C2-labeled adenine at this position in the twister ribozyme, which is significantly shifted compared to the pK a of the same adenine in the substrate alone. This finding pinpoints at a potential role for adenine-6 in the catalytic mechanism besides the previously identified invariant guanine-48 and a Mg 2+ ion, both of which are directly coordinated to the non-bridging oxygen atoms of the scissile phosphate; for the latter, additional evidence stems from the observation that Mn 2+ or Cd 2+ accelerated cleavage of phosphorothioate substrates. The relevance of this metal ion binding site is further emphasized by a new 2.6 Å X-ray structure of a 2′-OCH 3 -U5 modified twister ribozyme. Keywordsmetal ion rescue; nucleoside modifications; oligoribonucleotides; perturbed pK a ; solid-phase synthesis Small self-cleaving ribozymes are widely distributed in nature [1] and are essential for rolling-circle-based replication of satellite RNAs. [2,3] Among them, the hepatitis delta virus (HDV) ribozyme [4][5][6][7][8] employs a divalent cation in the active site for catalysis, while the remaining small self-cleaving ribozymes including hammerhead, [2,9,10] hairpin, [3,[11][12][13] glmS, [14][15][16] and Varkud Satellite [17] employ principles of general acid-base and electrostatics for catalysis. Very recently, a new class of nucleolytic ribozymes (termed twister) has been discovered, [18] and soon thereafter, crystal structures were published that revealed a common double-pseudoknot overall architecture for the twister ribozyme but showed clear distinctions in residue and divalent cation alignments at the cleavage site. [19][20][21] While the O. sativa twister ribozyme was off-line orthogonally aligned with a fully base-paired stem P1, [19,20] the env22 twister ribozyme was in-line oriented at the cleavage step A6-U5, with a Mg 2+ coordinated to the scissile phosphate. Furthermore, for the env22 twister ribozyme, stem P1 formed only the two central base pairs (Figure 1) while the neighboring nucleotides U1 and U4 were instead engaged in stacked base triplet interactions (U4-A49-A34 and U1-A50-U33; Figure 1B). [21] Košutić et al.Page 2 Angew Chem Int Ed Engl. Author manuscript; available in PMC 2016 January 16. Author Manuscript Author ManuscriptAuthor Manuscript Author ManuscriptThese contrasting observations were the starting point for the present investigation. A thorough comparison of the two structures (PDB: 4OJI for O. sativa and PDB: 4RGF for env22) revealed that the conserved adenosine (A6; env22 numbering is used throughout) at the cleavage site adopts nearly identical conformations involving ex...

show abstract

Understanding the Acid–Base Properties of Adenosine: The Intrinsic Basicities of N1, N3 and N7

Cited by 71 publications

References 56 publications

Structural basis for the fast self-cleavage reaction catalyzed by the twister ribozyme

Structural basis for the fast self-cleavage reaction catalyzed by the twister ribozyme

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

A Mini‐Twister Variant and Impact of Residues/Cations on the Phosphodiester Cleavage of this Ribozyme Class

Contact Info

Product

Resources

About