Crystal Structure of Biotin Synthase, an S -Adenosylmethionine-Dependent Radical Enzyme

Abstract

The crystal structure of biotin synthase from Escherichia coli in complex with S-adenosyl-Lmethionine and dethiobiotin has been determined to 3.4 angstrom resolution. This structure addresses how "AdoMet radical" or "radical SAM" enzymes use Fe 4 S 4 clusters and S-adenosyl-L-methionine to generate organic radicals. Biotin synthase catalyzes the radical-mediated insertion of sulfur into dethiobiotin to form biotin. The structure places the substrates between the Fe 4 S 4 cluster, essential for radical generation, and the Fe 2 S 2 cluster, postulated to be the source of sulfur, with both clusters in unprecedented coordination environments.Biotin synthase (BioB) catalyzes the final step in the biotin biosynthetic pathway, the conversion of dethiobiotin (DTB) to biotin. This remarkable reaction uses organic radical chemistry for the insertion of a sulfur atom between nonactivated carbons C6 and C9 of DTB (Scheme 1). BioB is a member of the "AdoMet radical" or "radical SAM" superfamily, which is characterized by the presence of a conserved CxxxCxxC sequence motif (C, Cys; x, any amino acid) that coordinates an essential Fe 4 S 4 cluster, as well as by the use of S-adenosyl-Lmethionine (AdoMet or SAM) for radical generation (1-3). AdoMet radical enzymes act on a wide variety of biomolecules. For example, BioB and lipoyl-acyl carrier protein synthase (LipA) are involved in vitamin biosynthesis; lysine 2,3-aminomutase (LAM) facilitates the fermentation of lysine; class III ribonucleotide reductase (RNR) activase and pyruvate formatelyase (PFL) activase catalyze the formation of glycyl radicals in their respective target proteins; and spore photoproduct lyase repairs ultraviolet light-induced DNA damage.AdoMet has been referred to as the "poor man's adenosylcobalamin" (4) because of the ability of both cofactors to generate a highly reactive 5′-deoxyadenosyl radical (5′-dA·), formed through homolytic cleavage of a C-Co bond in the case of adenosylcobalamin (AdoCbl) and through reductive cleavage of a C-S bond in the case of AdoMet (5). In AdoMet radical enzymes, the formation of 5′-dA· requires the addition of one electron, provided in E. coli by reduced flavodoxin and transferred first into an Fe 4 S 4 cluster and then into AdoMet (3). In the reaction catalyzed by BioB, there is general agreement that 5′-dA· generated from AdoMet oxidizes DTB (6), but the number and types of FeS clusters and of other cofactors involved in the reaction have been a subject of controversy (7)(8)(9)(10)(11)(12)(13)(14). Protein preparation-dependent cofactor differences have led to two mechanistic proposals for the method of S insertion in BioB. One proposal involves the use of an Fe 2 S 2 cluster as the sulfur source for biotin, and is consistent with 34 S isotopic labeling studies (15) and with the observed destruction of an Fe 2 S 2 cluster

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

“…The crystal structure of BioB shows two possible monomer-monomer interface regions, consistent with either a dimer or tetramer, and the major interface is reported as the most likely dimer interface by Berkovitch, et al [6]. This dimer interface involves two N-terminal helices and helices 7 and 8 from the core (αβ) 8 barrel [6]. If loss of the [2Fe-2S] 2+ cluster were to significantly alter the barrel fold, then one consequence might be disruption of the dimer interface.…”

“…Although the gene sequence predicts a monomer molecular weight of 38.7 kDa, they report a native molecular weight of 104 kDa (n = 2.7) and conclude that the native protein is most likely a dimer. The crystal structure of BioB shows two possible monomer-monomer interface regions, consistent with either a dimer or tetramer, and the major interface is reported as the most likely dimer interface by Berkovitch, et al [6]. This dimer interface involves two N-terminal helices and helices 7 and 8 from the core (αβ) 8 barrel [6].…”

“…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]. The structure of E. coli BioB [6] shows that AdoMet is covalently coordinated to a unique iron in the catalytic [4Fe-4S] 2+ cluster, and both are bound to an extended loop at the C-terminal end of an (αβ) 8 barrel, with the [4Fe-4S] 2+ cluster ∼5-7 Å from the exterior of the protein. In contrast, the [2Fe-2S] 2+ cluster is bound within the core of the (αβ) 8 barrel, in close proximity to dethiobiotin and ∼15-20 Å from the surface of the protein.…”

“…When 57 Fe-labeled enzyme is analyzed by Mössbauer spectroscopy prior to and during turnover, the most notable changes are the conversion of a narrow quadropole doublet associated with the protein-bound [2Fe-2S] 2+ cluster to overlapping broad quadropole doublets associated with Fe 2+ ions coordinated to various buffer components [15,16]. Finally, the most compelling evidence comes from the crystal structure of BioB, which shows that a bridging sulfide of the [2Fe-2S] 2+ cluster is found in very close proximity to the dethiobiotin C9 methyl (∼4.7 Å) and there is no room for any other sulfur containing substrates to bind within the active site; this space is completely filled by the two substrates and the two FeS clusters [6].…”

“…In vivo, this process is may be mediated by the iron-sulfur cluster assembly systems (isc and/or suf operons in E. coli). However, the binding site for the [2Fe-2S] 2+ cluster is located deep within the (αβ) 8 barrel [6], >15 Å from solvent, and therefore access to this site could limit the cluster assembly process. Opening the BioB enzyme active site to facilitate reassembly of the [2Fe-2S] 2+ cluster would require either a significant conformational change or would require localized or global unfolding of the polypeptide chain, a process that might render the protein susceptible to degradation.…”