Characterization of the Thioredoxin System from Colwellia psychrerythraea and Thermosipho africanus

Abstract

The thioredoxin system is a ubiquitous oxidoreductase system that consists of the enzyme thioredoxin reductase, the protein thioredoxin and the cofactor nicotinamide adenine dinucleotide phosphate. The system has been comprehensively studied from many organisms, such as Escherichia coli; however, structural and functional analysis of this system from psychrophilic and thermophilic bacteria has not been as extensive. In this study, the thioredoxin system proteins of a psychrophilic bacterium, Colwellia psychrerythraea, and a thermophilic bacterium, Thermosipho africanus, were characterized using biophysical, biochemical and X-ray crystallography techniques. Analysis of the complete genome sequence of each of these thioredoxin systems suggested the presence of a putative thioredoxin reductase, at least three thioredoxin in Colwellia psychrerythraea, and at least two thioredoxin in Thermosipho africanus. In this study, the identified putative thioredoxin system components were cloned, overexpressed, purified and characterized. Our studies have indicated that the thioredoxin system proteins from Thermosipho africanus were more stable, able to withstand both higher temperatures and higher concentrations of guanidine hydrochloride before denaturing, than those from Escherichia coli, which in turn were more stable than those from Colwellia psychrerythraea. Consistent with these results, kinetic assays indicated that the thioredoxin reductase from Thermosipho africanus had a higher optimal temperature than that from Escherichia coli, which in turn had a higher optimal temperature than that from Colwellia psychrerythraea. To understand and identify the differences that may contribute to these results, X-ray crystallography was used to determine the structure of Thermosipho africanus thioredoxin 1 and thioredoxin reductase. The structure of thioredoxin 1 showed the typical thioredoxin fold, although lacked the N-terminal region that was found in Escherichia coli thioredoxin 1. The structure of thioredoxin reductase resembled closely that from Escherichia coli. Our studies have therefore identified a novel thermophilic and a novel psychrophilic thioredoxin system that structurally and functionally behave like the thioredoxin system from Escherichia coli.