Chemistry Weekly Seminar - Naheda Sahtout

Title:

Characterizing the Thioredoxin System from the Thermophilic Bacterium, Thermosipho africanus

Abstract:

The thioredoxin system is a ubiquitous oxidoreductase system that consists of the enzyme thioredoxin reductase (TrxR), the protein thioredoxin (Trx) and the cofactor nicotinamide adenine dinucleotide phosphate (NADPH). The system has been comprehensively studied from many organisms, such as Escherichia coli (E. coli); however, structural and functional analysis of this system from thermophilic bacteria has not been as extensive. Thermosipho africanus (T. africanus) is a thermophilic bacterium that was first isolated from a marine hydrothermal area in Djibouti, Africa. Analysis of the complete genome sequence of T. africanus strain TCF52B, suggested the presence of two putative Trxs (TaTrx1 and TaTrx2) and a TrxR (TaTrxR) as components of its thioredoxin system. In this study, TaTrx1 and TaTrxR have been successfully cloned, overexpressed and purified and characterized using biophysical techniques, biochemical assays and X-ray crystallography. Our studies have indicated, not surprisingly, that TaTrx1 and TaTrxR are far more stable than the thioredoxin system components of E. coli. These two proteins were able to withstand both higher temperatures and higher concentrations of guanidine hydrochloride before denaturing. Consistent with these results, kinetic assays indicated that TaTrxR had a higher optimal temperature (70 °C) for activity, compared to E. coli TrxR (EcTrxR, 55 °C). Furthermore, TaTrxR was found to be catalytically more efficient at its optimal temperature. To understand and identify the differences that may contribute to these results, X-ray crystallography was used to determine the structure of TaTrx1 and TaTrxR. Our results provide further insight into the mode of action of the thioredoxin system in extremophiles.