Probing riboswitch–ligand interactions using thiamine pyrophosphate analogues

Abstract

The Escherichia coli thiM riboswitch forms specific contacts with its natural ligand, thiamine pyrophosphate (TPP or thiamine diphosphate), allowing it to generate not only nanomolar binding affinity, but also a high degree of discrimination against similar small molecules. A range of synthetic TPP analogues have been used to probe each of the riboswitch-ligand interactions. The results show that the pyrimidine-sensing helix of thiM is exquisitely tuned to select for TPP by recognising the H-bonding donor and acceptors around its aminopyrimidine ring and also by forming π-stacking interactions that may be sensitive to the electronics of the ring. The central thiazolium ring of TPP appears to be more important for ligand recognition than previously thought. It may contribute to binding via long-range electrostatic interactions and/or by exerting an electron withdrawing effect on the pyrimidine ring, allowing its presence to be sensed indirectly and thereby allowing discrimination between thiamine (and its phosphate esters) and other aminopyrimidines found in vivo. The pyrophosphate moiety is essential for submicromolar binding affinity, but unexpectedly, it does not appear to be strictly necessary for modulation of gene expression.

Novel TPP-riboswitch activators bypass metabolic enzyme dependency

“…On the other hand, the addition of 500 μM TT ( 1 ) represses the expression of β–galactosidase expression to a level similar to that of thiamine. TT has been shown to depend on diphosphorylation to gain activity in vitro (Chen et al, 2012). Thus, we anticipate that TT is phosphorylated by the endogenous set of thiamine converting enzymes.…”

“…Due to its close structural similarity to the natural substrate thiamine, TT is likely to be recognized and phosphorylated by endogenous bacterial enzymes such as ThiK. Using ITC measurements Chen and colleagues revealed that the chemically synthesized, diphosphorylated form of TT , named triazolethiamine pyrophosphate (TTPP), binds to the thiM (Figure 4A) riboswitch with an affinity of 370 nM, whereas for the natural ligand TPP a K D of 8 nM was measured (Chen et al, 2012). Additionally, in vitro translation assays proved that binding of TTPP induces a change in riboswitch secondary structure to sequester the SD-sequence and inhibits efficient protein translation (Chen et al, 2012).…”

“…These findings are in line with the observations of Chen al. recently reporting that the diphosphorylated triazolethiamine indeed interacts with the thiM riboswitch in vitro , albeit with decreased affinity (Chen et al, 2012). We went a significant step further and demonstrated that these compounds are effective in E. coli and, more importantly, that their activity depends on proteins involved in the metabolic pathways of thiamine uptake and synthesis.…”

“…Others have shown that analogs with a positively charged middle ring bind much better to the thiM riboswitch than ones with a neutral ring at this position (Chen et al, 2012). It was suggested that this is due to the electron drawing effect of the middle ring, which enhances the binding of the pyrimidine moiety to the cognate part of the aptamer domain.…”

Thiamine and selected thiamine antivitamins — biological activity and methods of synthesis

“…First, as a cofactor of many enzymes which control bioenergetic [47–50], amino acid metabolism [51], and transformation of various carbohydrates, including pentoses, necessary for the synthesis of nucleotides [52]. Second, we cannot underestimate the non-coenzymatic role of phosphorylated derivatives of thiamine in control of cell metabolism by: allosteric regulation of enzymes [53,47–49], transmission of nerve signals in synapses and likely involved in signaling pathways associated with receiving stimuli from the environment [54,55], and regulation of protein synthesis by so-called riboswitches in microorganisms and plants [56,57]. Finally, the results of many studies strongly suggest that thiamine, its phosphorylated derivatives, and thiamine-dependent enzymes play an important role in the reaction of microorganisms [58,59], animals [60,61], and plants [2,62] on various environmental factors like oxidative stress and pathogens.…”