Details
| Original language | English |
|---|---|
| Pages (from-to) | 1363-1372 |
| Number of pages | 10 |
| Journal | Biological chemistry |
| Volume | 383 |
| Issue number | 9 |
| Publication status | Published - 1 Sept 2002 |
Abstract
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.
Keywords
- 3-mercaptopyruvate, Arabidopsis thaliana, Mutagenesis, Sulfurtransferase, Thiosulfate
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)
- Biochemistry
- Biochemistry, Genetics and Molecular Biology(all)
- Molecular Biology
- Biochemistry, Genetics and Molecular Biology(all)
- Clinical Biochemistry
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In: Biological chemistry, Vol. 383, No. 9, 01.09.2002, p. 1363-1372.
Research output: Contribution to journal › Article › Research › peer review
}
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 -