Identification of the [Fe-S] cluster-binding residues of Escherichia coli biotin synthase

The gene encoding Escherichia coil biotin synthase (bioB) has been expresse d as a histidine fusion protein, and the protein was purified in a single s tep using immobilized metal affinity chromatography, The His(6)-tagged prot ein was fully functional in in vitro and in vivo biotin production assays. Analysis of all the published bioB sequences identified a number of conserv ed residues. Single point mutations, to either serine or threonine, were ca rried out on the four conserved (Cys-53, Cys-57, Cys-60, and Cys-188) and o ne non-conserved (Cys-288) cysteine residues, and the purified mutant prote ins were tested both for ability to reconstitute the [2Fe-2S] clusters of t he native (oxidized) dimer and enzymatic activity. The C188S mutant was ins oluble. 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 wer e verified using electrospray mass spectrometry. The results indicate that the His(6) tag and the C288T mutation have no effect on the activity of bio tin synthase when compared with the wild-type protein. The C53S, C57S, and C60S mutant proteins, both as prepared and reconstituted, were unable to co vert 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] c luster binding, whereas Cys-188 plays a hitherto unknown structural role in biotin synthase.