Identification of the [Fe-S] Cluster-binding Residues of Escherichia coli Biotin Synthase

McIver, Lisa; Baxter, Robert L.; Campopiano, Dominic J.

doi:10.1074/jbc.275.18.13888

Cited by 29 publications

(32 citation statements)

References 45 publications

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“…The presence of a serine residue as a naturally occurring ligand for a protein bound [4Fe-4S] cluster has not been previously observed, but a serine for cysteine site directed mutation has often been used to modulate or eliminate cluster activity [46][47][48][49][50][51]. Examination of the electron density near the auxiliary cluster ( Figure 3B) shows clear density for the serine ligand (Ser283) with a short Fe-O bond length (1.8 Å in the MTA complex), which likely reflects deprotonation of the serine ligand.…”

Section: Discussionmentioning

confidence: 99%

“…This cluster is held in place by three conserved cysteine residues [14] and an unanticipated ligand from close to the C-terminus, Ser283. Serine is previously unreported as a natural ligand for [4Fe-4S] clusters, although this residue has been introduced as a cluster ligand by site directed mutagenesis [46][47][48][49][50][51]. In the TeLipA2:SAH complex structure, the Ser283 alkoxy oxygen atom lies in close proximity to an additional metal atom, which because of its abundance in the crystallization medium (initially approximately 160 mM), has been modeled as a sodium ion (Na-O distance 2.5 Å, Figure 3B).…”

Section: Structure Of Telipa2mentioning

confidence: 99%

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Structures of lipoyl synthase reveal a compact active site for controlling sequential sulfur insertion reactions

Harmer

Hiscox

Dinis

et al. 2014

Biochemical Journal

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Summary Statement: The biosynthesis of lipoyl cofactors requires two lipoyl synthase mediated sulfur insertions. We report the crystal structures of a lipoyl synthase complexed with S-adenosylhomocysteine or 5'-methylthioadenosine. Models based on these structures identify likely substrate binding sites.Keywords: radical SAM, cofactors, crystal structure, enzyme catalysis, sulfur Abbreviations used: LipA, lipoyl synthase; BioB, biotin synthase; SAM, Sadenosylmethionine; LCD, lipoyl carrier domain; MTA, 5'-methylthioadenosine; RS, radical SAM; ACP, acyl carrier protein; SsLipA, Sulfolobus solfataricus LipA; TeLipA2 Thermosynechococcus elongatus LipA2; Ec, Escherichia coli; 5'-dA, 5'-deoxyadenosine. 2 ABSTRACTLipoyl cofactors are essential for living organisms and are produced by the insertion of two sulfur atoms into the relatively unreactive C-H bonds of an octanoyl substrate. This reaction requires lipoyl synthase, a member of the radical SAM enzyme superfamily. Herein we present crystal structures of lipoyl synthase with two [4Fe-4S] clusters bound at opposite ends of the TIM barrel, the usual fold of the radical SAM superfamily. The cluster required for reductive SAM cleavage conserves the features of the radical SAM superfamily, but the auxiliary cluster is bound by a CX 4 CX 5 C motif unique to lipoyl synthase. The fourth ligand to the auxiliary cluster is an extremely unusual serine residue. Site directed mutants show this conserved serine ligand is essential for the sulfur insertion steps. One crystallized LipA complex contains MTA, a breakdown product of SAM, bound in the likely SAM binding site. Modelling has identified an 18 Å deep channel, well-proportioned to accommodate an octanoyl substrate. These results suggest the auxiliary cluster is the likely sulfur donor, but access to a sulfide ion for the second sulfur insertion reaction requires the loss of an iron atom from the auxiliary cluster, which the serine ligand may enabled.3

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Section: Discussionmentioning

confidence: 99%

Section: Structure Of Telipa2mentioning

confidence: 99%

Structures of lipoyl synthase reveal a compact active site for controlling sequential sulfur insertion reactions

Harmer

Hiscox

Dinis

et al. 2014

Biochemical Journal

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“…c Nanomoles of biotin formed/nmol of enzyme monomer (Ϯ0.04) under the conditions of the assay described previously (13). min) photo-illumination process.…”

Section: Adomet Oxidizes the Reduced [4fe-4s] 1ϩ Cluster Of Biob Intomentioning

confidence: 99%

“…Finally, studies of site-directed mutants have demonstrated the requirement of six cysteines for activity. Cys-53, Cys-57, and Cys-60 (numbers referred to the Escherichia coli enzyme) are thought to be important for chelation and function of the [4Fe-4S] cluster (13,15). The function of Cys-97, Cys-128, and Cys-188, which are also absolutely required, is still unknown (24).…”

mentioning

confidence: 99%

Reductive Cleavage of S-Adenosylmethionine by Biotin Synthase from Escherichia coli

Choudens

Sanakis

Hewitson³

et al. 2002

Journal of Biological Chemistry

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A new class of iron-sulfur enzymes has recently emerged. They are defined on the basis of their absolute requirement for S-adenosylmethionine (AdoMet) 1 (1). The combination of a reduced iron-sulfur cluster and AdoMet is generally considered to result in the reductive cleavage of AdoMet and the generation of a 5Ј-deoxyadenosyl radical essential for initiation of catalysis. The prototypes for this class of enzymes are the activating components of anaerobic ribonucleotide reductase (RNR) and pyruvate formate lyase (PFL), lysine aminomutase (LAM), and biotin synthase (BioB) (2-5). The proteins of these systems have been shown to assemble an oxygen-sensitive [4Fe-4S] 2ϩ cluster, probably chelated by the three cysteines in the CXXX-CXXC sequence that is conserved among all enzymes and a fourth, not yet identified ligand (6 -15). For RNR, PFL activases, and LAM, it has been shown that the cluster has to be reduced to the 1ϩ state before it can react with AdoMet (6, 16 -18). The reaction results in the production of one equivalent of methionine, one equivalent of 5Ј-deoxyadenosine, and approximately one equivalent of a substrate radical. The latter is a protein-bound glycyl radical in RNR and PFL and a lysinederived radical in LAM (19 -21). In all cases, electrons are not transferred from the cluster to AdoMet in the absence of the substrate, showing that the reaction is thermodynamically unfavorable. In this report we present studies of the same reaction for BioB.Biotin synthase (BioB) catalyzes the incorporation of a sulfur atom into dethiobiotin to form biotin (see Scheme 1 below) (22). In vitro, this reaction is very inefficient and the reported yields of biotin never exceed 1-2 equivalents per protein after several hours reaction, raising the possibility that BioB is not an enzyme but a reactant (23-25). The protein from Escherichia coli is a 76-kDa homodimer that binds a [4Fe-4S] center on each polypeptide chain (14,26). Recently, Jarrett and co-workers (27) suggested that the active form of BioB contained two clusters, one [4Fe-4S] and one [2Fe-2S], per polypeptide chain. On the other hand, using a different procedure for reconstitution of iron centers in BioB, we are able to generate enzyme of similar activity with no more than 4 Fe and 4 S atoms per monomer mainly in the form of a [4Fe-4S] cluster (14,15).In addition to an Fe-S cluster and AdoMet, several proteins and cofactors are necessary for biotin formation: namely NADPH, DTT, iron, sulfide, cysteine, flavodoxin, and flavodoxin reductase (23,24). The flavodoxin system is thought to effect in the reduction of the cluster. Requirement of iron, sulfide, and DTT suggests that the cluster is labile and has to be reconstituted during catalysis. Finally, the source of the sulfur for the biosynthesis of dethiobiotin has not been firmly established, although it has been suggested to be the cluster itself (28). This function is proposed to specifically reside in the [2Fe-2S] cluster in Jarrett's enzyme preparations (25). It should be noted that, in vivo, the sulfu...

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“…Indeed, it is evolved to accommodate simultaneously two different oxygen-sensitive clusters, one 2ϩ and one 2ϩ per monomer, with unique roles in catalysis (6). The [4Fe-4S] could be ligated by three cysteines of a CX 3 CX 2 C box, and a fourth, still unidentified ligand (7)(8)(9). More recently, Ugulava et al (10) proposed that the [2Fe-2S] center could be ligated at an alternative site not previously occupied by the [4Fe-4S] center.…”

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confidence: 99%

The Plant Biotin Synthase Reaction

Picciocchi¹,

Douce²,

Alban

2003

Journal of Biological Chemistry

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In plants, the last step of the biotin biosynthetic pathway is localized in mitochondria. This chemically complex reaction is catalyzed by the biotin synthase protein, encoded by the bio2 gene in Arabidopsis thaliana. Unidentified mitochondrial proteins in addition to the bio2 gene product are obligatory for the reaction to occur. In order to identify these additional proteins, potato mitochondrial matrix was fractionated onto different successive chromatographic columns. Combination experiments using purified Bio2 protein and the resulting mitochondrial matrix subfractions together with a genomic based research allowed us to identify mitochondrial adrenodoxin, adrenodoxin reductase, and cysteine desulfurase (Nfs1) proteins as essential components for the plant biotin synthase reaction. Arabidopsis cDNAs encoding these proteins were cloned, and the corresponding proteins were expressed in Escherichia coli cells and purified. Purified recombinant adrenodoxin and adrenodoxin reductase proteins formed in vitro an efficient low potential electron transfer chain that interacted with the bio2 gene product to reconstitute a functional plant biotin synthase complex. Bio2 from Arabidopsis is the first identified protein partner for this specific plant mitochondrial redox chain.

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Identification of the [Fe-S] Cluster-binding Residues of Escherichia coli Biotin Synthase

Cited by 29 publications

References 45 publications

Structures of lipoyl synthase reveal a compact active site for controlling sequential sulfur insertion reactions

Structures of lipoyl synthase reveal a compact active site for controlling sequential sulfur insertion reactions

Reductive Cleavage of S-Adenosylmethionine by Biotin Synthase from Escherichia coli

The Plant Biotin Synthase Reaction

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