Enzyme 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... 

The vitamin biotin is formed in nature (left) by condensation of L-alanine with pimeloyl-CoAto form 8-amino-7-oxononanoate (AON). This compound is seen at the upper left of the center structure joined as a Schiff base with the coenzyme pyridoxal phosphate (PLP). This is a product complex of the enzyme AON synthase (see Webster et ah, Biochemistry 39,516-528,2000) Courtesy of D. Alexeev,... 

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]. 

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). ... 

Some vitamins undetfio a rather unique transformation prior to becoming functional. They are covalently attached to specific enzymes. Biotin, for example, is covalently bound to the biotin-requiring enzymes. Pantothenic acid, in a modified form, is covalently bound to fatty acid synthase. Riboflavin, following conversion to thecofaclor form, is bound to succinate dehydrogenase, as well as to a few other enzymes requiring riboflavin-based cofactors. 

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]. 

Biotin synthases from several organisms have been purified and characterized, the first one from E. coliP They are indeed [Fe-S] proteins, isolated as homodimers containing one [2Fe-2S] center per monomer when purified aerobically. However, the elucidation of the nature of the [Fe-S] centers in the active enzyme has represented a very difficult problem, as will be discussed below. 

For several years, she studied chemical reaction mechanisms, especially those involving sulfoxide carbanions and their synthetic applications. Then, she turned to mechanistic enzymology. Her main interests concerned steroid isomerases and cytochrome P-450, vitamin K-dependent carboxylations, and biotin biosynthesis. She contributed to the mechanistic understanding of several enzymes of the pathway, namely, amino-oxopelargo-nate synthase, diaminopelargonate aminotransferase, and more importantly biotin synthase. 

Biotin synthase (BioB) and lipoate synthase were two other enzymes that, prior to the identification of the superfamily, were shown to contain iron-sulfur clusters and to utilize SAM for catalyzing the radical-mediated insertion of sulfur into unactivated C-H bonds."" ... 

In conclusion, iron and sulfide are assembled in the small protein of the anaerobic ribonucleotide reductase primarily as one [2Fe-2S] + center per polypeptide chain. Our data suggest that imder the strong reducing conditions required for radical-ization and activation of the enzyme, two centers are converted into one [4Fe-4S] cluster at the interface of the two polypeptide chains. Such a cluster dimerization has been recently also observed in the case of the FNR transcription factor protein [49] and in the case of biotin synthase [50] (Scheme 10-4). 

GTP = 5 -guanosine triphosphate AE = Activating enzyme BAN = Backbone amide nitrogen BioB = Biotin synthase CD = Circular dichroism cyt = Cytochrome DFT = Density functional theory DMSO = Dimethylsulfoxide Dx = Desulforedoxin ENDOR = Electron-nuclear double resonance EPR = Electron paramagnetic resonance ESEEM = Electron-spin echo envelop modulation ETF = Electron transferring flavoprotein EXAFS = Extended x-ray absorption fine structure FAD = Flavin adenine dinucleotide Fd = Ferredoxin FMN = Flavin mononucleotide FNR = Fumarate-nitrate reduction FTIR =... 

Figure 8 Reactions catalyzed by the radical-SAM superfamily. A. Lysine 2,3-aminomutase. B. Activating enzymes. C. Biotin synthase. D. Lipoic acid synthase. E. Spore photoproduct lyase.

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