NifS-like proteins are cysteine desulphurases required for the mobilization of sulphur from cysteine. They are present in all organisms, where they are involved in iron–sulphur (Fe–S) cluster biosynthesis. In eukaryotes, these enzymes are present in mitochondria, which are the major site for Fe–S cluster assembly. The genome of the model plant Arabidopsis thaliana contains two putative NifS-like proteins. A cDNA corresponding to one of them was cloned by reverse-transcription PCR, and named AtNFS2. The corresponding transcript is expressed in many plant tissues. It encodes a protein highly related (75% similarity) to the slr0077-gene product from Synechocystis PCC 6803, and is predicted to be targeted to plastids. Indeed, a chimaeric AtNFS2–GFP fusion protein, containing one-third of AtNFS2 from its N-terminal end, was addressed to chloroplasts. Overproduction in Escherichia coli and purification of recombinant AtNFS2 protein enabled one to demonstrate that it bears a pyridoxal 5′-phosphate-dependent cysteine desulphurase activity in vitro, thus being the first NifS homologue characterized to date in plants. The putative physiological functions of this gene are discussed, including the attractive hypothesis of a possible role in Fe–S cluster assembly in plastids.
Abbreviations used: CHX, cycloheximide; DTT, dithiothreitol; Fe–S, iron–sulphur; GFP, green fluorescent protein; NLS, nuclear localization sequence; NTA, nitrilotriacetate; OPA, o-phthalaldehyde; ORF, open reading frame; PEG, poly(ethylene glycol); PLP, pyridoxal 5′-phosphate; RT-PCR, reverse-transcription PCR.
The AtNFS2 cDNA sequence has been deposited in the GenBank®, EMBL, DDBJ and GSDB Nucleotide Sequence Databases under the accession number AY078068.