Enzyme-mediated sulfide production for the reconstitution of [2Fe-2S] clusters into apo-biotin synthase of Escherichia coli. Sulfide transfer from cysteine to biotin.
We previously showed that biotin synthase in which the (Fe-S) cluster was labelled with 34S by reconstitution donates 34S to biotin [B. Tse Sum Bui, D. Florentin, F. Fournier, O. Ploux, A. Méjean & A. Marquet (1998) FEBS Lett. 440, 226-230]. We therefore proposed that the source of sulfur was very likely the (Fe-S) centre. This depletion of sulfur from the cluster during enzymatic reaction could explain the absence of turnover of the enzyme which means that to restore a catalytic activity, the clusters have to be regenerated. In this report, we show that the NifS protein from Azotobacter vinelandii and C-DES from Synechocystis as well as rhodanese from bovine liver can mobilize the sulfur, respectively, from cysteine and thiosulfate for the formation of a [2Fe-2S] cluster in the apoprotein of Escherichia coli biotin synthase. The reconstituted enzymes were as active as the native enzyme. When [35S]cysteine was used during the reconstitution experiments in the presence of NifS, labelled (Fe35S) biotin synthase was obtained. This enzyme produced [35S]biotin, confirming the results obtained with the 34S-reconstituted enzyme. NifS was also effective in mobilizing selenium from selenocystine to produce an (Fe-Se) cluster. However, though NifS could efficiently reconstitute holobiotin synthase from the apoform, starting from cysteine, these two effectors had no significant effect on the turnover of the enzyme in the in vitro assay.
Bui
B T
BT
Laboratoire de Chimie Organique Biologique-CNRS UMR 7613, Université Paris VI, France.
Escalettes
F
F
Chottard
G
G
Florentin
D
D
Marquet
A
A
D001426:Bacterial Proteins / Q000378:metabolism; D001710:Biotin / Q000378:metabolism*; D003545:Cysteine / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D007506:Iron-Sulfur Proteins / Q000378:metabolism*; D013440:Sulfides / Q000378:metabolism*; D013466:Sulfurtransferases / Q000378:metabolism*
D001426:Bacterial Proteins; D007506:Iron-Sulfur Proteins; D013440:Sulfides; C080001:nifS protein, Bacteria; D001710:Biotin; D013466:Sulfurtransferases; C057985:biotin synthetase; D003545:Cysteine