Characterization of a sulfurtransferase from Arabidopsis thaliana

A database search for similarities between sequenced parts of the Arabidops is thaliana genome with known sulfurtransferase sequences from Escherichia coli and mammals was undertaken to obtain information about plant sulfurtra nsferase-like proteins. One gene and several homologous EST clones were ide ntified. One of the EST clones was used for screening an Arabidopsis cDNA l ibrary. 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 o rganisms 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-li ke isozymes in Arabidopsis. Recombinant proteins with and without the putat ive transit peptide were expressed in E. coli with an N-terminal His(6)-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 defin ition; however, both recombinant proteins preferred 3-mercaptopyruvate to t hiosulfate. A monospecific antibody produced by using the mature recombinan t protein as an antigen recognized a single protein band in total extracts of Arabidopsis plants equating to the full-length protein size. A single ba nd equating to the size of the mature protein was detected from purified Ar abidopsis mitochondria, but there was no antigenic reaction with any protei n from chloroplasts. The function of the protein is still speculative. Now tools are available to elucidate the roles and substrates of this sulfurtra nsferase in higher plants.