Biotin-synthase

Using a combination of techniques such as EPR, resonance Raman, and MCD spectroscopy, the conversion of [2Fe-2S] into [4Fe—4S] centers has been found to take place under reducing conditions in E. coli biotin synthase 281). The as-prepared form of this enzyme has been thought to contain one [2Fe-2S] center per monomer, coordinated by the three cysteine residues of the motif Cys-X3-Cys-X2-Cys and by a fourth, noncysteinyl ligand. Upon reduction, a [4Fe-4S] cluster bridging two monomers may be formed in the active enzyme. In the reduced state, the [4Fe-4S] center is characterized by a mixture of S = I and S = k spin states giving EPR features at g 5.6 and... 

Bui BTS, Florentin D, Fournier F, et al. 1998. Biotin synthase mechanism on the origin of sulphur. FEBS Fett 440 226-30. 

Gibson KJ, Pelletier DA,Turner IM Sr. 1999. Transfer of snlfnr to biotin from biotin synthase (bioB protein). Biochem Biophys Res Commnn 254 632-5. 

Another interesting cluster conversion is the joining of two Fe2S2 clusters in a protein to form a single Fe4S4 cluster at the interface between a dimeric protein. Such a cluster is present in the nitrogenase iron protein (Fig. 24-2) and probably also in biotin synthase.294 The clusters in such proteins can also be split to release the monomers. 

This compound undergoes a two-step ATP-dependent cyclization352-355 to form dethiobiotin. The final step, insertion of sulfur into dethiobiotin, is catalyzed by biotin synthase, a free-radical-dependent enzyme related to pyruvate formate lyase (Fig. 15-16). It transfers the sulfur from cysteine via an Fe-S cluster.3553 Biosynthesis of lipoic acid involves a similar insertion of two sulfur atoms into octanoic acid.356 See also p. 1410. 

So far, two types of aminomutase have been investigated in detail. Lysine 2,3-aminomutase from Clostridium subterminale SB4 is the example par excellence for the SAM-dependent type of aminomutase. Several other enzymes belonging to the same family are known. Examples are biotin synthase [82], pyruvate formate lyase [83, 84], and anaerobic ribonucleotide reductase [85]. 

Figure 13. Transformation of dethiobiotin to biotin by biotin synthase. Working hypothesis adapted from reference 43.

Figure 11.3. Biosynthesis of biotin. Keto-aminopelargonic acid synthase, EC 2.3.1.47 diaminopelargonic acid synthase (aminotransferase), EC 2.6.1.62 dethiobiotin synthase, EC 6.3.3.3 and biotin synthase, EC 2.8.1.6.

The final reaction, catalyzed by biotin synthase, involves the insertion of sulfur between the unreactive methyl and methylene carbons of dethiobiotin. The enzyme has an iron-sulfur box, and requires NADPff and a ferredoxin or flavo-doxin reducing system. S-Adenosylmethionine is also required, and is cleaved to yield methionine and a 5 -deoxyadenosyl radical during the reaction. Biotin synthase is a member of the radical SAM family of enzymes, in which the catalytic 5 -deoxyadenosyl radical is formed from S-adenosylmethionine,... 

It is possibly incorrect to consider biotin synthase an enzyme in the true sense of the word it has a turnover number of 1. It only catalyzes the synthesis of a single molecule of biotin from dethiohiotin before being inactivated. This is because the iron-sulfur cluster of the protein is the source of the sulfur that is incorporated into biotin. There is some evidence that the enzyme can be reactivated by incorporation of sulfur from cysteine, but in vitro addition of the enzymes believed to catalyze this reaction has no effect on the turnover number of the enzyme (Frey, 2001 Marquet et al., 2001). 

The [Fe4S4(LS3)(SR )] cluster has been shown to engage in an electrophilic attack of the sulfonium ion, while causing reductive cleavage of the cofactor S-adenosylmethionine. This behavior is analogous to the enzymatic action of biotin synthase and other enzymes in the S-adenosylmethionine family see Iron-Sulfur Proteins). ... 

Biotin (vitamin H, 6, Figs. 1 and 10) acts a cofactor of carboxylases. It can be produced in bacteria, plants, and some fungi (46). The biosynthetic pathway involves four steps that start from alanine (78) and pimeoyl-CoA (79). Carboxylation and cyclization of 81 affords dethiobiotin (82), which is then converted into biotin (6) by the iron/sulfur protein, biotin synthase, in an unusual radical mechanism (47). 

DTB to 1 is effected [40-45]. A lack of data owing to low catalytic activity of the enzyme, i.e., biotin synthase, which Is responsible for the last step, obscures elucidation of the mechanism. A major byproduct of the fermentation of 1 is, in most cases, DTB. The difficulty to obtain an enzymatic system with high biotin synthase activity makes the fermentation approach to 1 mostly impractical for the commercial production [46-49]. 

This very interesting reaction involves the formal insertion of a sulfur atom into two unactivated CH bonds. The purified protein contains a [2Fe-2S] cluster [131]. A defined system, consisting of biotin synthase, flavodoxin, llavodoxin reductase, fructose 1,6-bisphosphate, cysteine, DTT, NADPH, ferrous ion, and SAM, capable of catalyzing the conversion of dethiobiotin to biotin has been characterized [132-134]. This system is still incomplete and gives a maximum of two moles of biotin per mole of biotin synthase. 

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