Poster abstracts

Poster number 86 submitted by Govardhan reddy Veerareddygari

The initial steady state kinetic characterization of a thermophilic pseudouridine synthase (Thermotoga maritima TruB)

Govardhan reddy Veerareddygari (Department of Chemistry, University of Louisville), Eugene G. Mueller (Department of Chemistry, University of Louisville)

Abstract:
Pseudouridine synthases (Ψ synthases) catalyze the isomerization of uridine (U) in RNA to pseudouridine (Ψ). Ψ synthases are classified into six different families based on sequence alignments and have a universally conserved aspartic acid residue that is essential for activity. RNA containing 5-fluorouridine ([F⁵U]RNA) has been used as a mechanistic probe. E. coli TruB (EcTruB) is not inhibited upon incubation with [F⁵U]RNA but instead handles it as a substrate. Thermotoga maritima TruB (TmTruB) on the other hand, is irreversibly inhibited by [F⁵U]RNA and forms an apparently covalent adduct with it. Despite this difference in behavior, cocrystals of [F⁵U]RNA and both EcTruB and TmTruB show a noncovalent complex in which the F⁵U is not only hydrated but rearranged to the C-glycoside isomer. This hydrated product was thought to result from the hydrolysis of an ester linkage between the active site Asp and C6 of the pyrimidine ring of F⁵U in agreement with one of the two proposed mechanisms for Ψ synthases. Later, the [¹⁸O] labeling studies on TruA, TruB and RluA, conducted by the Mueller group proved that the oxygen atom in the hydrated products of F⁵U comes from the solvent, rather than from the active site Asp. The interesting difference in behavior of a thermophilic versus mesophilic TruB towards [F⁵U]RNA demands the characterization of TmTruB and extension of mechanistic studies that were performed on its mesophilic counterpart. The initial steady state kinetic characterization of TmTruB using a new HPLC based assay is complete, and the groundwork has been laid for the mechanistic studies of TmTruB with [F⁵U]RNA.

Keywords: pseudouridine, fluorouridine