Biotin Synthase: Purification, Characterization as a [2Fe-2S]Cluster Protein, and in vitro Activity of the Escherichia coli bioB Gene Product

Abstract

We report here the first purification of the protein encoded by the Escherichia coli bioB gene. One species of this protein runs on native gels with an electrophoretic mobility typical of a protein with m = 82 kDa, suggesting the protein is a dimer (gene sequence predicts m = 38.7 kDa). There are two iron- and two acid-labile sulfur atoms per protein monomer. Solutions containing the protein are red and have an absorbance spectrum characteristic of proteins with [2Fe-2S] clusters. In its oxidized native state, the protein is EPR-silent. Upon addition of dithionite, the protein's UV-visible absorbance spectrum is very slowly bleached, and an EPR active species is produced that displays a signal at gavg = 1.95. All these results are consistent with this protein containing one [2Fe-2S] cluster per monomer. The EPR spin quantitation is only 5-10% of expected. Since this protein loses iron upon reduction with dithionite, the low-spin quantitation is probably due to cluster instability in the reduced state. Another species of the bioB gene products has also been purified which runs on native gels with an electrophoretic mobility typical of a protein with m = 104 kDa. This species appears to be a dimer with one [2Fe-2S] cluster per dimer. The 104-kDa protein can be converted to the 82-kDa protein upon incubation with Fe3+ and S2-. The bioB gene product we have isolated is active in the conversion of dethiobiotin to biotin in vitro in the presence of NADPH, AdoMet, Fe3+ or Fe2+, and additional unidentified factors from the crude extracts of E. coli. The Km for dethiobiotin in this reaction has been found to be 2 microM.

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

“…Preparation of Apo and Holo Wild-type Biotin Synthase and Mutants-This was carried out using methods previously described (14,20).…”

“…Whereas the active sites have been proposed to involve the cysteine thiols of the common GXCXXXCXXCXQ motif as a "cysteine ([Fe-S] coordination) box" motif, there has been as yet no experimental evidence to support this contention (20). In this paper we describe studies on mutants of the biotin synthase protein that uniquely identify the key protein cysteine residues involved in formation of the [Fe-S] cluster.…”

“…The major native form of the core biotin synthase, however, appears to be a 76-kDa dimer, each monomer containing an [2Fe-2S] cluster (20). Spectroscopic studies suggest that reduction of the two [2Fe-2S] clusters in the oxidized protein dimer, an obligatory step in the mechanism, results in formation of a biotin synthase dimer containing a single [4Fe-4S] cluster in which each of the iron centers is coordinated to a thiol ligand(s) of the protein (21)(22)(23).…”

Probing the NADPH-binding site of Escherichia coli flavodoxin oxidoreductase

“…The Escherichia coli flavodoxin-NADP + oxidoreductase (FLDR, or flavodoxin reductase, EC 1.18.1.2) is a 27.6 kDa FADcontaining enzyme which interacts with both E. coli flavodoxin (FLD) and ferredoxin redox partners and participates in a variety of important electron-transport systems in the bacterium ; including the cobalamin-dependent methionine synthase and biotin synthase reactions [1,2]. In addition, the FLDR\FLD system is used to support the function of pyruvate-formate lyase and ribonucleotide reductase, which are key enzymes for the anaerobic growth of E. coli [3,4].…”

Loss of iron–sulfur clusters from biotin synthase as a result of catalysis promotes unfolding and degradation

“…The enzyme is a homodimeric iron-sulfur protein in which each monomer contains both a [4Fe-4S] 2+ cluster and a [2Fe-2S] 2+ cluster [2]. The [4Fe-4S] 2+/+ cluster is bound to a conserved CxxxCxxC motif that is common to a large superfamily of AdoMet-dependent radical enzymes, while the [2Fe-2S] 2+ cluster, found only in BioB, is bound to 3 cysteines and an arginine that are located throughout the primary sequence [3][4][5].…”