Direct Observation of Multiple Tautomers of Oxythiamine and their Recognition by the Thiamine Pyrophosphate Riboswitch

Abstract

Structural diversification of canonical nucleic acid bases and nucleotide analogues by tautomerism has been proposed to be a powerful on/off switching mechanism allowing regulation of many biological processes mediated by RNA enzymes and aptamers. Despite the suspected biological importance of tautomerism, attempts to observe minor tautomeric forms in nucleic acid or hybrid nucleic acid-ligand complexes have met with challenges due to the lack of sensitive methods. Here, a combination of spectroscopic, biochemical and computational tools probed tautomerism in the context of an RNA aptamer-ligand complex; studies involved a model ligand, oxythiamine pyrophosphate (OxyTPP), bound to the thiamine pyrophosphate (TPP) riboswitch (an RNA aptamer) as well as its unbound non-phosphorylated form, oxythiamine (OxyT). OxyTPP, like canonical heteroaromatic nucleic acid bases, has a pyrimidine ring that forms hydrogen bonding interactions with the riboswitch. Tautomerism was established using two-dimensional infrared (2D IR) spectroscopy, variable temperature FTIR and NMR spectroscopies, binding isotope effects (BIEs) and computational methods. All three possible tautomers of OxyT, including the minor enol tautomer, were directly identified and their distributions were quantitated. In the bound form, BIE data suggested that OxyTPP existed as a 4'-keto tautomer that was likely protonated at the N1'-position. These results also provide a mechanistic framework for understanding the activation of riboswitch in response to deamination of the active form of vitamin B1 (or TPP). The combination of methods reported here revealing the fine details of tautomerism can be applied to other systems where the importance of tautomerism is suspected.

Tautomerism provides a molecular explanation for the mutagenic properties of the anti-HIV nucleoside 5-aza-5,6-dihydro-2′-deoxycytidine

“…Because minor tautomers (e.g., enol or imino) are typically rare, obtaining direct evidence to support their existence has been a challenge to experimentalists. The present study, by using a battery of complementary spectroscopic tools (41), has shown that KP1212, unlike canonical nucleic acid bases, exists as an ensemble of up to five tautomers in solution. The data further show that KP1212 displays a strong propensity to exist in an enol form, rather than the classical keto form characteristic of nucleic acid bases.…”

Role of tautomerism in RNA biochemistry

“…Tautomeric equilibria are influenced by chemical and physical factors including the presence of metals, temperature (Peng et al 2013;Singh et al 2014) and pH (CS Peng, V Singh, BI Fedeles, D Li, T Amariuta, JM Essigmann, and A Tokmakoff, in prep.). The effect of metals on nucleobase tautomerism has been reviewed previously (Lippert and Gupta 2009), while the influence of temperature on tautomeric equilibria is discussed later in this review.…”

“…4, 5, below;Cochrane and Strobel 2008a). Tautomerism has also been proposed to influence recognition of xanthine and OxyTPP by the purine and TPP riboswitches, respectively (Thore et al 2008;Gilbert et al 2009;Singh et al 2014). Although not discussed below, tautomeric preference of DNA and RNA polymerases is another area where formation of minor tautomeric forms may have important physiological consequences; specifically, the minor tautomeric forms of canonical nucleobases have been long speculated to induce spontaneous mutations, both during DNA replication (Watson and Crick 1953;Topal and Fresco 1976;Bebenek et al 2011;Wang et al 2011) and during transcription (Shugar and Kierdaszuk 1985).…”

“…Tautomerism has also been invoked as a molecular rationale for the interaction of certain base analogs with RNA aptamers. For example, the purine riboswitch (Gilbert et al 2009), and the thiamine pyrophosphate (TPP) riboswitch (Thore et al 2008;Gilbert et al 2009;Singh et al 2014) are proposed to bind to the minor tautomeric forms of their nonnative ligands, xanthine, and oxythiamine pyrophosphate (OxyTPP), respectively.…”

“…Challenges of studying tautomerism in aqueous conditions include the fast rates of the tautomeric equilibria, the low abundance of minor tautomers, and the high chemical and structural similarity, between the corresponding minor and major tautomeric species. Traditional spectroscopic methods, although sensitive to certain chemical and structural properties, have been of limited use for studying tautomerism under aqueous conditions (Peng and Tokmakoff 2012;Singh et al 2014). However, recent important developments in spectroscopic methods are beginning to overcome historical limitations and are starting to allow comprehensive characterization of tautomeric equilibria of base analogs free in solution or in the context of nucleic acids .…”