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J Biol Chem, Vol. 275, Issue 18, 13888-13894, May 5, 2000

Identification of the [Fe-S] Cluster-binding Residues of Escherichia coli Biotin Synthase*

Lisa McIverDagger , Robert L. Baxter§, and Dominic J. Campopiano§

From the 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

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 His6-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 His6 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.

* This work was supported in part by the Biotechnology and Biological Sciences Research Council (BBSRC).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequences reported in this paper have been submitted to the Swiss Protein Database under Swiss-Prot accession numbers P12996, O67104, P54967, P19206, P53557, P46396, Q9Z6L5, Q47862, P44987, O25956, P94966, P46715, O06601, P32451, O60050, P36569, and P73538.

Dagger Supported by the Department of Chemistry, Edinburgh University.

§ To whom correspondence should be addressed. Tel.: 44 131 650 4712; Fax: 44 131 650 7155; E-mail: Dominic.Campopiano@ed.ac.uk (for D. J. C.) or Tel.: 44 131 650 4708; Fax: 44 131 650 7155; E-mail: r.baxter@ed.ac.uk (for R. L. B.).

Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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