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M. Burow, D. Kessler, J. Papenbrock
Enzymatic Activity of the Arabidopsis Sulfurtransferase Resides in the C-Terminal Domain But Is Boosted by the N-Terminal Domain and the Linker Peptide in the Full- Length Enzyme
Sulfurtransferases/rhodaneses are a group of enzymes widely distributed in plants, animals, and bacteria that catalyze the transfer of sulfur from a donor molecule to a thiophilic acceptor substrate. Sulfurtransferases (STs) consist of two globular domains of nearly identical size and conformation connected by a short linker sequence. In plant STs this linker sequence is exceptionally longer than in sequences from other species. The Arabidopsis ST1 protein (AJ131404) contains five cysteine residues: one residue is universally conserved in all STs and considered to be catalytically essential; a second one, closely located in the primary sequence, is conserved only in sequences from eukaryotic species. Of the remaining three cysteine residues two are conserved in the so far known plant STs and one is unique to the Arabidopsis ST1. The aim of our study was to investigate the role of the twodomain structure, of the unique plant linker sequence and of each cysteine residue. The N and Cterminal domains of the Arabidopsis ST1, the fulllength protein with a shortened linker sequence and several pointmutated proteins were overexpressed in E. coli, purified and used for enzyme activity measurements. The Cterminal domain itself displayed ST activity which could be increased by adding the separately prepared Nterminal domain. The activity of an ST1 derivative with a shortened linker sequence was reduced by more than 60% of the wildtype activity, probably because of a drastically reduced protein stability. The replacement of each cysteine residue resulted in mutant forms which differed significantly in their stability, in the specific ST activities, and in their kinetic parameters which were determined for 3-mercaptopyruvate as well as thiosulfate as sulfur substrates: mutation of the putative active site cysteine (C332) essentially abolished activity; for C339 a crucial role at least for the turnover of thiosulfate could be identified.
Biological Chemistry, Walter de Gruyter
Print ISSN: 1431-6730
Volume: 383, 09/2002
Pages: 1363 - 1372