Abstract
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.
Highlights
The biotin operon of Escherichia coli contains six open reading frames that encode at least four of the proteins essential for the conversion of pimeloyl-CoA to biotin [1]
There is no doubt that addition of extracts derived from E. coli bioBϪ strains can enhance activity of biotin synthase, but whether this is due to the presence of a thiamine pyrophosphate-stabilized protein [7], high levels of constitutive low molecular weight cellular components such as fructose 1,6-biphosphate and the labile AdoMet-derived product of the 7,8-diaminononanoate synthase reaction [5], or to a combination of several of these factors is still open to question
The intermediacy of 9-mercaptodethiobiotin, racemization at C-9 [10] (but not at C-6 [11]), and the requirement for at least two molecules of AdoMet for each ring formation step provides clues to the mechanism of C–S bond formation [12]. Taken together these results suggest that the mechanism involves the following: (a) initial AdoMet-dependent radical formation at C-9; (b) capture of sulfur by the C-9 radical to form a 9-mercaptodethiobiotin derivative; and (c) a second AdoMet-initiated radical formation by abstraction of the pro-S hydrogen at C-6 to generate a conformationally restrained radical that subsequently captures the C-9 thiol resulting in tetrahydrothiophene ring clo
Summary
Materials—Electrophoresis was carried out using a Bio-Rad Protean II minigel system (protein) and a Life Technologies, Inc., H5 system (DNA). The deep red fractions containing biotin synthase eluted at 300 mM salt These were combined, brought to a final concentration of 10% ammonium sulfate, and loaded onto a phenyl-Sepharose hydrophobic interaction column (1 ϫ 30 cm) that had been equilibrated with buffer B containing ammonium sulfate (10%). Mass Spectrometry—Prior to mass spectrometry all protein samples were passed through an Aquapure reverse phase C-4 column at a constant trifluoroacetic acid concentration of 0.1% using a linear gradient of 10 –100% acetonitrile in water over 40 min at a flow rate of 1 ml/min and the separation monitored at 280 nm. A typical assay mixture contained biotin synthase or mutant protein (5 M), potassium chloride (10 mM), dethiobiotin (50 M), AdoMet (150 M), Fe(NH4)2(SO4) (5 mM), NADPH (1 mM), fructose 1,6-biphosphate (5 mM), L-cysteine (500 M), DTT (10 mM), PCOi cell-free extract (100 l), FLD (12.5 M), and FLDR (2 M). Labile sulfide analysis was determined as previously reported [20]
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