Characterization of a sulfurtransferase from Arabidopsis thaliana.
A database search for similarities between sequenced parts of the Arabidopsis thaliana genome with known sulfurtransferase sequences from Escherichia coli and mammals was undertaken to obtain information about plant sulfurtransferase-like proteins. One gene and several homologous EST clones were identified. One of the EST clones was used for screening an Arabidopsis cDNA library. The isolated full-length clone consists of 1134 bp and encodes a 42.6 kDa protein that includes a putative transit peptide sequence of about 7.1 kDa. Sequence comparisons with known sulfurtransferases from different organisms confirmed high homology between them and the existence of several highly conserved regions. Results of a Southern blot performed with genomic Arabidopsis DNA showed the occurrence of at least two sulfurtransferase-like isozymes in Arabidopsis. Recombinant proteins with and without the putative transit peptide were expressed in E. coli with an N-terminal His6-tag, purified by affinity chromatography and tested for enzyme activity using different sulfur donors and acceptors. Both recombinant proteins catalyzed the formation of SCN- from thiosulfate and cyanide as a rhodanese per definition; however, both recombinant proteins preferred 3-mercaptopyruvate to thiosulfate. A monospecific antibody produced by using the mature recombinant protein as an antigen recognized a single protein band in total extracts of Arabidopsis plants equating to the full-length protein size. A single band equating to the size of the mature protein was detected from purified Arabidopsis mitochondria, but there was no antigenic reaction with any protein from chloroplasts. The function of the protein is still speculative. Now tools are available to elucidate the roles and substrates of this sulfurtransferase in higher plants.
Papenbrock
J
J
Institute for Botany, University of Hannover, Germany. Jutta.Papenbrock@mbox.botanik.uni-hannover.de
Schmidt
A
A
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000166:cytology / Q000201:enzymology*; D015139:Blotting, Southern; D002736:Chloroplasts / Q000201:enzymology; D003001:Cloning, Molecular; D003486:Cyanides / Q000378:metabolism; D003545:Cysteine / Q000031:analogs & derivatives* / Q000378:metabolism; D020224:Expressed Sequence Tags; D017343:Genes, Plant / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008928:Mitochondria / Q000201:enzymology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010802:Phylogeny; D021382:Protein Sorting Signals / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017384:Sequence Deletion / Q000235:genetics; D013379:Substrate Specificity; D013466:Sulfurtransferases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013884:Thiosulfate Sulfurtransferase / Q000737:chemistry / Q000235:genetics; D013885:Thiosulfates / Q000378:metabolism*
D003486:Cyanides; D007527:Isoenzymes; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D013885:Thiosulfates; C008366:3-mercaptopyruvic acid; D013466:Sulfurtransferases; D013884:Thiosulfate Sulfurtransferase; D003545:Cysteine