TY - JOUR

T1 - 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

AU - Burow, Meike

AU - Kessler, Dorothea

AU - Papenbrock, Jutta

N1 - Funding information: We would like to thank P. von Trzebiatowski and J. Volker for their excellent technical assistance, and M. Bauer for the gel-exclusion experiment. Many thanks to Dr. J. Meens, University of Hannover, for sequencing work and to Prof. H. Bisswanger, University of Tübingen, for valuable suggestions concerning enzyme kinetics. We are grateful to Prof. A. Schmidt, University of Hannover, for helpful discussions. The work was financially supported by the Deutsche Forschungsgemeinschaft (PA 764/1-1, PA 764/1-2) and the Fonds der Chemischen Industrie.

PY - 2002/9/1

Y1 - 2002/9/1

N2 - 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 two-domain structure, of the unique plant linker sequence and of each cysteine residue. The N- and C-terminal domains of the Arabidopsis ST1, the full-length protein with a shortened linker sequence and several point-mutated proteins were overexpressed in E. coli, purified and used for enzyme activity measurements. The C-terminal domain itself displayed ST activity which could be increased by adding the separately prepared N-terminal domain. The activity of an ST1 derivative with a shortened linker sequence was reduced by more than 60% of the wild-type 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.

AB - 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 two-domain structure, of the unique plant linker sequence and of each cysteine residue. The N- and C-terminal domains of the Arabidopsis ST1, the full-length protein with a shortened linker sequence and several point-mutated proteins were overexpressed in E. coli, purified and used for enzyme activity measurements. The C-terminal domain itself displayed ST activity which could be increased by adding the separately prepared N-terminal domain. The activity of an ST1 derivative with a shortened linker sequence was reduced by more than 60% of the wild-type 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.

KW - 3-mercaptopyruvate

KW - Arabidopsis thaliana

KW - Mutagenesis

KW - Sulfurtransferase

KW - Thiosulfate

UR - http://www.scopus.com/inward/record.url?scp=0036754012&partnerID=8YFLogxK

U2 - 10.1515/BC.2002.155

DO - 10.1515/BC.2002.155

M3 - Article

C2 - 12437129

AN - SCOPUS:0036754012

VL - 383

SP - 1363

EP - 1372

JO - Biological chemistry

JF - Biological chemistry

SN - 1431-6730

IS - 9

ER -