Identification of the [Fe-S] cluster-binding residues of Escherichia coli biotin synthase.
The gene encoding Escherichia coli biotin synthase (bioB) has been expressed as a histidine fusion protein, and the protein was purified in a single step using immobilized metal affinity chromatography. The His(6)-tagged protein was fully functional in in vitro and in vivo biotin production assays. Analysis of all the published bioB sequences identified a number of conserved residues. Single point mutations, to either serine or threonine, were carried out on the four conserved (Cys-53, Cys-57, Cys-60, and Cys-188) and one non-conserved (Cys-288) cysteine residues, and the purified mutant proteins were tested both for ability to reconstitute the [2Fe-2S] clusters of the native (oxidized) dimer and enzymatic activity. The C188S mutant was insoluble. The wild-type and four of the mutant proteins were characterized by UV-visible spectroscopy, metal and sulfide analysis, and both in vitro and in vivo biotin production assays. The molecular masses of all proteins were verified using electrospray mass spectrometry. The results indicate that the His(6) tag and the C288T mutation have no effect on the activity of biotin synthase when compared with the wild-type protein. The C53S, C57S, and C60S mutant proteins, both as prepared and reconstituted, were unable to covert dethiobiotin to biotin in vitro and in vivo. We conclude that three of the conserved cysteine residues (Cys-53, Cys-57, and Cys-60), all of which lie in the highly conserved "cysteine box" motif, are crucial for [Fe-S] cluster binding, whereas Cys-188 plays a hitherto unknown structural role in biotin synthase.
McIver
L
L
Edinburgh Centre for Protein Technology, Department of Chemistry, Joseph Black Building, the University of Edinburgh, The King's Buildings, West Mains Road, Edinburgh EH9 3JJ, Scotland, United Kingdom.
Baxter
R L
RL
Campopiano
D J
DJ
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D001710:Biotin / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D008969:Molecular Sequence Data; D009876:Operon / Q000235:genetics; D016415:Sequence Alignment; D013379:Substrate Specificity; D013466:Sulfurtransferases / Q000235:genetics* / Q000378:metabolism
D001426:Bacterial Proteins; D001710:Biotin; D013466:Sulfurtransferases; C057985:biotin synthetase