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Biochemical Society Transactions (2005) Volume 33, part 4
Biotin synthase mechanism: an overview M. Lotierzo, B. Tse Sum Bui, D. Florentin, F. Escalettes and A. Marquet1 Synthese, ` Structure et Fonction de Molecules ´ Bioactives, UMR CNRS 7613, Universite´ Paris VI, 4 place Jussieu, 75252 Paris Cedex 05, France
Abstract Biotin synthase, a member of the ‘radical SAM’ (S-adenosylmethionine) family, converts DTB (dethiobiotin) into biotin. The active form of the Escherichia coli enzyme contains two (Fe-S) centres, a (4Fe-4S) and a (2Fe-2S). The (4Fe-4S)2+/+ mediates the electron transfer required for the reductive cleavage of SAM into methionine and a DOA• (deoxyadenosyl radical). Two DOA• , i.e. two SAM molecules, are consumed to activate the positions 6 and 9 of DTB. A direct transfer of isotope from the labelled substrate into DOAH (deoxyadenosine) has been observed with 2 H, although not quantitatively, but not with tritium. The source of the sulphur introduced to form biotin is still under debate. We have shown that the (2Fe-2S)2+ cluster can be reconstituted in the apoenzyme with S2− and Fe2+ . When S2− was replaced by [34 S2− ], [35 S2− ] or Se2− , biotin containing mostly the sulphur isotopes or selenium was obtained. This leads us to favour the hypothesis that the (2Fe-2S) centre is the sulphur donor, which may explain the absence of turnover of the enzyme. DTBSH (9-mercaptodethiobiotin), which already contains the sulphur atom of biotin, was shown to be an alternative substrate of biotin synthase both in vivo and with a crude extract. When this compound was tested with a well-defined in vitro system, the same turnover of one and similar reaction rates were observed for DTB and DTBSH. We postulate that the same intermediate is formed from both substrates.
Introduction The ‘radical SAM’ (S-adenosylmethionine) family of enzymes, now recognized as a very large group [1] represent a fascinating, only recently explored field of enzymology. They catalyse different reactions, which always involve a chemically difficult process, requiring radical chemistry. They share a common mechanistic step, namely the production of a DOA• (deoxyadenosyl radical) generated by the monoelectronic reduction of SAM followed by the homolytic cleavage of a C–H bond by DOA• [2–4]. They all contain a (4Fe-4S)2+,1+ cluster liganded by three cysteines which belong to a perfectly conserved CX3 CX2 C motif representing the signature of the family. This (Fe-S) centre is the final electron donor to SAM. Before the discovery of this superfamily, three enzymes using SAM as a source of DOA• were already characterized: lysine 2,3-aminomutase [5], pyruvate formate lyase activase [6] and anaerobic ribonucleotide reductase activase [7]. Biotin synthase, the enzyme responsible for the conversion of DTB (dethiobiotin) into biotin, came to be the fourth candidate, when it was shown that (i) SAM was absolutely required to obtain an active cell-free system [8,9], and (ii) SAM was not the sulphur donor [10]. A plausible mechanistic scheme was rapidly proposed (Scheme 1) [11,12]. In Escherichia coli, the involvement of NADPH, flavodoxin [13] and flavodoxin reductase [14] as the electron transfer system was established, and the site of Key words: biotin synthase mechanism, (Fe-S) clusters, hydrogen transfer mechanism, ‘radical SAM’ family, sulphur source, turnover. Abbreviations used: DOA• , deoxyadenosyl radical; DOAH, deoxyadenosine; DTB, dethiobiotin; 9-DTBSH, 9-mercaptodethiobiotin; SAM, S-adenosylmethionine. 1 To whom correspondence should be addressed (email
[email protected]).
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the first C–H bond cleavage, postulated at C-9 of DTB, was deduced from previous results obtained in our group [10,15]. An in vitro assay performed under anaerobic conditions, containing the electron transfer system (NADPH, flavodoxin, flavodoxin reductase), the as-isolated enzyme, SAM, the substrate DTB, Fe2+ , S2− and dithiothreitol was set up. Very surprisingly, the turnover observed under these conditions was at most one. Of course, many questions had to be answered in order to validate the proposed mechanism. If many points have now been elucidated, through the efforts of different groups, several questions still remain.
The nature of the (Fe-S) clusters in the active form of biotin synthase The nature of the (Fe-S) clusters in the active form of the enzyme is a difficult problem, which gave rise to long debates. There is now more or less agreement among three groups, those of J.J. Jarrett, M.K. Johnson and ours, that the active form of biotin synthase contains two clusters, a (4Fe-4S)2+,1+ common to all ‘radical SAM’ enzymes, which mediates the electron transfer, and a (2Fe-2S)2+ [16–18], located at a different site [19]. This conclusion is supported by biochemical experiments, electron paramagnetic resonance, ¨ resonance Raman and Mossbauer spectroscopies, and the recently solved 3D structure [20]. The (4Fe-4S) centre is highly oxygen sensitive [21,22], and the as-isolated protein contains uniquely the (2Fe-2S)2+ cluster. We have also found, studying whole cells overexpressing biotin synthase by ¨ Mossbauer spectroscopy, that the two clusters were present, although in different proportions [23].
Coenzymology: biochemistry of vitamin biogenesis and cofactor-containing enzymes
Scheme 1 Proposed mechanism for the formation of biotin from DTB and 9-DTBSH
The same in vitro activity was obtained starting with a reconstituted biotin synthase containing a (4Fe-4S)2+ and a (2Fe-2S)2+ centre [18,24], or the as-isolated form containing only the (2Fe-2S)2+ cluster, provided Fe2+ and S2− were ¨ added to the assay [25,26]. We have studied by Mossbauer spectroscopy, under strict anaerobic conditions, an enzyme preparation containing essentially the (2Fe-2S)2+ form, as well as Fe2+ and S2− : the (4Fe-4S)2+ centre was spontaneously reconstituted at the expense of Fe2+ and S2− , and also of (2Fe2S)2+ centre, the final stage being the 1/1 mixture of two clusters [17]. Starting with the apo form, the same activity was observed [26]. These consistent results show that the clusters are easily reconstituted in their active configuration. Whatever the conditions, the same maximum turnover of one was obtained.
The formation of radicals at C-6 and C-9 of DTB Three aspects have been considered regarding the formation of radicals at C-6 and C-9 of DTB. (a) The first question concerns the cleavage of SAM, the occurrence of which was not obvious by considering the respective redox potentials of a (4Fe-4S)2+,1+ and of sulfonium salts. It is now proved that in all the studied ‘radical SAM’ enzymes, SAM is the fourth ligand of the (4Fe-4S) centre, which is liganded by only three cysteines. This situation facilitates electron transfer [2–4,27]. (b) How many SAM molecules, i.e. DOA• radicals, are needed to activate the two carbon atoms of DTB? We have established that about 2.6 ± 0.2 mol of DOAH (deoxyadenosine) were produced per mol of biotin synthesized. This was determined by HPLC analysis (D. Florentin, H. Vilcou,
M. Lotierzo, B. Tse Sum Bui and A. Marquet, unpublished work), as well as using SAM tritiated in the adenine moiety. With SAM tritiated in the sulphonium methyl group, we could verify that methionine and DOAH were produced, in stoichiometric amounts [28]. This value greater than two is consistent with the use of two SAMs, but also reveals some abortive consumption of DOA• . However, other data allow us to conclude that indeed, two SAMs are required [29]. (c) Is there a direct transfer of hydrogen between the substrate DTB and DOAH? It is well known that the ‘radical SAM’ family contains two subfamilies: in the first one, the hydrogen removed from the substrate is directly transferred into DOAH. In the second one, DOA• , generated by the activase, abstracts a hydrogen from a glycine residue of the enzyme and it is a protein radical which is responsible for reaction with the substrate [30]. It had to be determined to which subfamily biotin synthase belonged. To do this, we synthesized DTB labelled with 2 H at different positions: [9-2 H3 ]DTB, [6-2 H2 ,9-2 H3 ]DTB, [6-2 HR ,9-2 H1 ]DTB and [6-2 HS ,9-2 H1 ]DTB. DOAH isolated from reactions with these differently labelled substrates contained 2 H. This established that biotin synthase catalyses the direct transfer of H• between the substrate and DOA• [29]. This is consistent with the absence in the sequence of a conserved glycine found in the second subfamily [30]. However, intriguingly, the amount of 2 H incorporated into DOAH was in each case lower (approx. 50%) than the expected amount, assuming that two DOA• are used to activate the C-9 and C-6 positions. In order to clarify this point, we prepared [9-3 H]DTB, since it should be easier to localize the label with tritium. Using this substrate, we found that the isolated DOAH contained only traces of tritium. Biotin specific activity was, C 2005
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as expected, approx. 2/3 of that of DTB [31,32], and we checked that the protein fraction did not contain any tritium. Radioactivity was found in water, but it was difficult to quantify (D. Florentin, H. Vilcou, M. Lotierzo, B. Tse Sum Bui and A. Marquet, unpublished work). This reveals that the reaction of DOA• with the substrate is more complex than expected. A satisfying explanation remains to be found.
Figure 1 Biotin formation from DTB (䊉) and 9-DTBSH (䊏) as a function of substrate concentration (A) and time at saturating concentrations (B)
The sulphur source The sulphur source remains a controversial point. Early studies have shown that biotin synthase itself was the sulphur source: we have shown that biotin was produced in a simplified assay containing only the enzyme, SAM, the substrate and dithiothreitol, with photoreduced deazaflavin as electron source [33], whereas Gibson et al. showed that 35 S-labelled biotin synthase purified from E. coli cells grown on [35 S]cysteine produced [35 S]biotin [34]. A possible hypothesis was that the (Fe-S) centre of the enzyme was the sulphur source. Indeed, reconstitution of the (2Fe-2S)2+ centre in the apoenzyme using 34 S2− or 35 2− S gave rise to a protein with a labelled (Fe-S)centre, which after purification produced labelled biotin in the in vitro assay [25,35]. We have recently produced biotin synthase containing a (2Fe-2Se)2+ centre, by reconstituting the apoprotein with Na2 Se instead of Na2 S. When the assay was performed in the presence of additional Na2 Se, selenobiotin was obtained without any trace of biotin (B. Tse Sum Bui, T. Maltioli and A. Marquet, unpublished work). At the time of the experiments with 34 S or 35 S reconstituted biotin synthase [25,35], the role of complex (Fe-S) centres was not yet elucidated. The finding that the active form of biotin synthase contains both (4Fe-4S) and (2Fe-2S) centres led to a consistent picture, with the (4Fe-4S) cluster responsible for the electron transfer, and the (2Fe-2S) cluster as sulphur source. In these experiments, the yield in biotin was rather low since no external sulphur source was added in the in vitro assay, in order to avoid sulphide exchanges. We now know that without the addition of S2− , the (4Fe-4S) centre cannot be fully reconstituted. The Xray structure of the mixed cluster form containing SAM and DTB revealed that the methyl group of DTB, which is proposed to react with the (2Fe-2S) centre, is properly oriented, at a compatible distance [20]. Another experimental ¨ confirmation came from Mossbauer experiments, which have shown that during the formation of biotin, the amount of (2Fe-2S) decreased, whereas the amount of (4Fe-4S) remained constant [17,24,36]. A different scenario involving a persulphide, formed on a cysteine residue of biotin synthase by cysteine desulphuration, has been proposed [37]. However, the experimental results supporting this hypothesis could not be reproduced by other groups [18,26]. On the other hand, the precise way the sulphur atom could be transferred from the (2Fe-2S)2+ cluster to the DTB radical requires clarification. We propose that the destruction of this (Fe-S) cluster is responsible for the absence of turnover, the enzyme being C 2005
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at the same time a substrate of the reaction. Whereas the maximal activity is obtained by adding all the cofactors to the apoenzyme, addition of fresh cofactors after one turn produced only a small additional amount of biotin (0.4 mol/ monomer), showing that the (2Fe-2S)2+ centre cannot be properly reconstituted. This led us to postulate that the site of this centre remained occupied by its degradation product and attempts to remove it were unsuccessful [26]. An independent experiment was performed using 9DTBSH (9-mercaptodethiobiotin) as substrate. This compound, which proved to be transformed into biotin in vivo [15] or in a cell-free system [10], was tested for the first time with purified enzymes in a well-defined assay. The main result is that even with this substrate, the turnover is also one, the same as with DTB (Figure 1). One could have expected a different behaviour since the sulphur atom is already present in this compound. The apparent K m value of 9-DTBSH is about 20 times higher than that of DTB (Figure 1A) but both substrates exhibit similar kinetics (Figure 1B) [26]. These results are consistent with the proposed mechanism if we assume that the same intermediate is involved (I in Scheme 1). In that case, there is no reason to expect similar K m values for both substrates. On the other hand, the similarity of the rates may be rationalized if the last step, the cyclization on C-6, is the rate-determining one.
Coenzymology: biochemistry of vitamin biogenesis and cofactor-containing enzymes
Conclusions In spite of significant progress, the mechanism of biotin synthase is not yet completely elucidated. We propose a selfconsistent hypothesis, but the way the hydrogen is transferred from the substrate to DOA• , and the way the substrate radical at C-9 of DTB reacts with sulphur awaits clarification. Our hypothesis accounts for the absence of in vitro turnover. Whether there is a turnover in vivo, and which are the partners involved in that case, has still to be established. This research has been supported in part by the European Union’s Human Potential Programme under Contract HPRN-CT-2002-00244 (Viteomics). M.L. acknowledges the financial support provided through this programme.
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