SMART MODE:

NORMAL GENOMIC

• Dinis P et al.
• X-ray crystallographic and EPR spectroscopic analysis of HydG, a maturase in [FeFe]-hydrogenase H-cluster assembly.
• Proc Natl Acad Sci U S A. 2015; 112: 1362-7
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• Hydrogenases use complex metal cofactors to catalyze the reversible formation of hydrogen. In [FeFe]-hydrogenases, the H-cluster cofactor includes a diiron subcluster containing azadithiolate, three CO, and two CN(-) ligands. During the assembly of the H cluster, the radical S-adenosyl methionine (SAM) enzyme HydG lyses the substrate tyrosine to yield the diatomic ligands. These diatomic products form an enzyme-bound Fe(CO)x(CN)y synthon that serves as a precursor for eventual H-cluster assembly. To further elucidate the mechanism of this complex reaction, we report the crystal structure and EPR analysis of HydG. At one end of the HydG (betaalpha)8 triosephosphate isomerase (TIM) barrel, a canonical [4Fe-4S] cluster binds SAM in close proximity to the proposed tyrosine binding site. At the opposite end of the active-site cavity, the structure reveals the auxiliary Fe-S cluster in two states: one monomer contains a [4Fe-5S] cluster, and the other monomer contains a [5Fe-5S] cluster consisting of a [4Fe-4S] cubane bridged by a mu2-sulfide ion to a mononuclear Fe(2+) center. This fifth iron is held in place by a single highly conserved protein-derived ligand: histidine 265. EPR analysis confirms the presence of the [5Fe-5S] cluster, which on incubation with cyanide, undergoes loss of the labile iron to yield a [4Fe-4S] cluster. We hypothesize that the labile iron of the [5Fe-5S] cluster is the site of Fe(CO)x(CN)y synthon formation and that the limited bonding between this iron and HydG may facilitate transfer of the intact synthon to its cognate acceptor for subsequent H-cluster assembly.

• Broderick JB, Duffus BR, Duschene KS, Shepard EM
• Radical S-adenosylmethionine enzymes.
• Chem Rev. 2014; 114: 4229-317

• Atta M et al.
• The methylthiolation reaction mediated by the Radical-SAM enzymes.
• Biochim Biophys Acta. 2012; 1824: 1223-30
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• Over the past 10 years, considerable progress has been made in our understanding of the mechanistic enzymology of the Radical-SAM enzymes. It is now clear that these enzymes appear to be involved in a remarkably wide range of chemically challenging reactions. This review article highlights mechanistic and structural aspects of the methylthiotransferases (MTTases) sub-class of the Radical-SAM enzymes. The mechanism of methylthio insertion, now observed to be performed by three different enzymes is an exciting unsolved problem. This article is part of a Special Issue entitled: Radical SAM enzymes and Radical Enzymology.

• Lin S, Cronan JE
• Closing in on complete pathways of biotin biosynthesis.
• Mol Biosyst. 2011; 7: 1811-21
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• Biotin is an enzyme cofactor indispensable to metabolic fixation of carbon dioxide in all three domains of life. Although the catalytic and physiological roles of biotin have been well characterized, the biosynthesis of biotin remains to be fully elucidated. Studies in microbes suggest a two-stage biosynthetic pathway in which a pimelate moiety is synthesized and used to begin assembly of the biotin bicyclic ring structure. The enzymes involved in the bicyclic ring assembly have been studied extensively. In contrast the synthesis of pimelate, a seven carbon alpha,omega-dicarboxylate, has long been an enigma. Support for two different routes of pimelate synthesis has recently been obtained in Escherichia coli and Bacillus subtilis. The E. coli BioC-BioH pathway employs a methylation and demethylation strategy to allow elongation of a temporarily disguised malonate moiety to a pimelate moiety by the fatty acid synthetic enzymes whereas the B. subtilis BioI-BioW pathway utilizes oxidative cleavage of fatty acyl chains. Both pathways produce the pimelate thioester precursor essential for the first step in assembly of the fused rings of biotin. The enzymatic mechanisms and biochemical strategies of these pimelate synthesis models will be discussed in this review.

• Lotierzo M, Bui BT, Leech HK, Warren MJ, Marquet A, Rigby SE
• Iron-sulfur cluster dynamics in biotin synthase: a new [2Fe-2S](1+) cluster.
• Biochem Biophys Res Commun. 2009; 381: 487-90
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• Biotin synthase (BioB) catalyses the final step in the biosynthesis of biotin. Aerobically purified biotin synthase contains one [2Fe-2S](2+) cluster per monomer. However, active BioB contains in addition a [4Fe-4S](2+) cluster which can be formed either by reconstitution with iron and sulfide, or on reduction with sodium dithionite. Here, we use EPR spectroscopy to show that mutations in the conserved YNHNLD sequence of Escherichia coli BioB affect the formation and stability of the [4Fe-4S](1+) cluster on reduction with dithionite and report the observation of a new [2Fe-2S](1+) cluster. These results serve to illustrate the dynamic nature of iron-sulfur clusters in biotin synthase and the role played by the protein in cluster interconversion.

• Boyd JM, Sondelski JL, Downs DM
• Bacterial ApbC protein has two biochemical activities that are required for in vivo function.
• J Biol Chem. 2009; 284: 110-8
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• The ApbC protein has been shown previously to bind and rapidly transfer iron-sulfur ([Fe-S]) clusters to an apoprotein (Boyd, J. M., Pierik, A. J., Netz, D. J., Lill, R., and Downs, D. M. (2008) Biochemistry 47, 8195-8202. This study utilized both in vivo and in vitro assays to examine the function of variant ApbC proteins. The in vivo assays assessed the ability of ApbC proteins to function in pathways with low and high demand for [Fe-S] cluster proteins. Variant ApbC proteins were purified and assayed for the ability to hydrolyze ATP, bind [Fe-S] cluster, and transfer [Fe-S] cluster. This study details the first kinetic analysis of ATP hydrolysis for a member of the ParA subfamily of "deviant" Walker A proteins. Moreover, this study details the first functional analysis of mutant variants of the ever expanding family of ApbC/Nbp35 [Fe-S] cluster biosynthetic proteins. The results herein show that ApbC protein needs ATPase activity and the ability to bind and rapidly transfer [Fe-S] clusters for in vivo function.

• Reyda MR, Dippold R, Dotson ME, Jarrett JT
• Loss of iron-sulfur clusters from biotin synthase as a result of catalysis promotes unfolding and degradation.
• Arch Biochem Biophys. 2008; 471: 32-41
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• Biotin synthase (BioB) is an S-adenosylmethionine radical enzyme that catalyzes addition of sulfur to dethiobiotin to form the biotin thiophane ring. In vitro, Escherichia coli BioB is active for only one turnover, during which the [2Fe-2S]2+ cluster is destroyed, one sulfide from the cluster is incorporated as the biotin thiophane sulfur, while Fe2+ ions and the remaining S2- ion are released from the protein. The present work examines the fate of the protein following the loss of the FeS clusters. We examine the quaternary structure and thermal stability of active and inactive states of BioB, and find that loss of either the [4Fe-4S]2+ or [2Fe-2S]2+ clusters results in destabilization but not global unfolding of BioB. Using susceptibility to limited proteolysis as a guide, we find that specific regions of the protein appear to be transiently unfolded following loss of these clusters. We also examine the in vivo degradation of biotin synthase during growth in low-iron minimal media and find that BioB is degraded by an apparent ATP-dependent proteolysis mechanism that sequentially cleaves small fragments starting at the C-terminus. BioB appears to be resistant to degradation and capable of multiple turnovers only under high-iron conditions that favor repair of the FeS clusters, a process most likely mediated by the Isc or Suf iron-sulfur cluster assembly systems.

• Bonomi F, Iametti S, Morleo A, Ta D, Vickery LE
• Studies on the mechanism of catalysis of iron-sulfur cluster transfer from IscU[2Fe2S] by HscA/HscB chaperones.
• Biochemistry. 2008; 47: 12795-801
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• The HscA/HscB chaperone/cochaperone system accelerates transfer of iron-sulfur clusters from the FeS-scaffold protein IscU (IscU(2)[2Fe2S], holo-IscU) to acceptor proteins in an ATP-dependent manner. We have employed visible region circular dichroism (CD) measurements to monitor chaperone-catalyzed cluster transfer from holo-IscU to apoferredoxin and to investigate chaperone-induced changes in properties of the IscU(2)[2Fe2S] cluster. HscA-mediated acceleration of [2Fe2S] cluster transfer exhibited an absolute requirement for both HscB and ATP. A mutant form of HscA lacking ATPase activity, HscA(T212V), was unable to accelerate cluster transfer, suggesting that ATP hydrolysis and conformational changes accompanying the ATP (T-state) to ADP (R-state) transition in the HscA chaperone are required for catalysis. Addition of HscA and HscB to IscU(2)[2Fe2S] did not affect the properties of the [2Fe2S] cluster, but subsequent addition of ATP was found to cause a transient change of the visible region CD spectrum, indicating distortion of the IscU-bound cluster. The dependence of the rate of decay of the observed CD change on ATP concentration and the lack of an effect of the HscA(T212V) mutant were consistent with conformational changes in the cluster coupled to ATP hydrolysis by HscA. Experiments carried out under conditions with limiting concentrations of HscA, HscB, and ATP further showed that formation of a 1:1:1 HscA-HscB-IscU(2)[2Fe2S] complex and a single ATP hydrolysis step are sufficient to elicit the full effect of the chaperones on the [2Fe2S] cluster. These results suggest that acceleration of iron-sulfur cluster transfer involves a structural change in the IscU(2)[2Fe2S] complex during the T --> R transition of HscA accompanying ATP hydrolysis.

• Marelja Z, Stocklein W, Nimtz M, Leimkuhler S
• A novel role for human Nfs1 in the cytoplasm: Nfs1 acts as a sulfur donor for MOCS3, a protein involved in molybdenum cofactor biosynthesis.
• J Biol Chem. 2008; 283: 25178-85
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• The human MOCS3 gene encodes a protein involved in activation and sulfuration of the C terminus of MOCS2A, the smaller subunit of the molybdopterin (MPT) synthase. MPT synthase catalyzes the formation of the dithiolene group of MPT that is required for the coordination of the molybdenum atom in the last step of molybdenum cofactor (Moco) biosynthesis. The two-domain protein MOCS3 catalyzes both the adenylation and the subsequent generation of a thiocarboxylate group at the C terminus of MOCS2A by its C-terminal rhodanese-like domain (RLD). The low activity of MOCS3-RLD with thiosulfate as sulfur donor and detailed mutagenesis studies showed that thiosulfate is most likely not the physiological sulfur source for Moco biosynthesis in eukaryotes. It was suggested that an L-cysteine desulfurase might be involved in the sulfuration of MOCS3 in vivo. In this report, we investigated the involvement of the human L-cysteine desulfurase Nfs1 in sulfur transfer to MOCS3-RLD. A variant of Nfs1 was purified in conjunction with Isd11 in a heterologous expression system in Escherichia coli, and the kinetic parameters of the purified protein were determined. By studying direct protein-protein interactions, we were able to show that Nfs1 interacted specifically with MOCS3-RLD and that sulfur is transferred from L-cysteine to MOCS3-RLD via an Nfs1-bound persulfide intermediate. Because MOCS3 was shown to be located in the cytosol, our results suggest that cytosolic Nfs1 has an important role in sulfur transfer for the biosynthesis of Moco.

• Zeng J, Zhao W, Liu Y, Xia L, Liu J, Qiu G
• Expression, purification and characterization of an iron-sulfur cluster assembly protein, IscU, from Acidithiobacillus ferrooxidans.
• Biotechnol Lett. 2007; 29: 1965-72
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• An iron-sulfur cluster assembly protein, IscU, is encoded by the operon iscSUA in Acidithiobacillus ferrooxidans. The gene of IscU was cloned and expressed in Escherichia coli. The protein was purified by one-step affinity chromatography to homogeneity. The protein was in apo-form, the [Fe(2)S(2)] cluster could be assembled in apoIscU with Fe(2+) and sulfide in vitro, and in the presence of IscA and IscS, the IscU could utilize L: -cysteine and Fe(2+) to synthesize [Fe(2)S(2)] cluster in the protein. Site-directed mutagenesis for the protein revealed that Cys37, Asp39, Cys63 and Cys106 were involved in ligating with the [Fe(2)S(2)] cluster.

• Hernandez HL et al.
• MiaB, a bifunctional radical-S-adenosylmethionine enzyme involved in the thiolation and methylation of tRNA, contains two essential [4Fe-4S] clusters.
• Biochemistry. 2007; 46: 5140-7
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• The radical-S-adenosylmethionine (radical-AdoMet) enzyme MiaB catalyzes the posttranscriptional methylthiolation of N-6-isopentenyladenosine in tRNAs. Spectroscopic and analytical studies of the reconstituted wild-type and C150/154/157A triple variant forms of Thermotoga maritima MiaB have revealed the presence of two distinct [4Fe-4S](2+,1+) clusters in the protein. One is coordinated by the three conserved cysteines in the radical-AdoMet motif (Cys150, Cys154, and Cys157) as previously reported, and the other, here observed for the first time, is proposed to be coordinated by the three N-terminal conserved cysteines (Cys10, Cys46, and Cys79). The two [4Fe-4S]2+ clusters have similar UV-visible absorption, resonance Raman, and Mossbauer properties but differ in terms of redox properties and the EPR properties of the reduced [4Fe-4S]1+ clusters. Reconstituted forms of MiaB containing two [4Fe-4S] clusters are more active than previously reported. Comparison of MiaB with other radical-AdoMet enzymes involved in thiolation reactions, such as biotin synthase and lipoate synthase, is discussed as well as a possible role of the second cluster as a sacrificial S-donor in the MiaB-catalyzed reaction.

• Abdel-Hamid AM, Cronan JE
• In vivo resolution of conflicting in vitro results: synthesis of biotin from dethiobiotin does not require pyridoxal phosphate.
• Chem Biol. 2007; 14: 1215-20
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• The source of the biotin sulfur atom remains a contested point in studies of biotin synthase (BioB) in vitro. Recent reports that BioB has an intrinsic pyridoxal phosphate (PLP)-dependent cysteine desulfurase activity were tested by depleting Escherichia coli cells of PLP. The BioB-dependent conversion of dethiobiotin to biotin proceeded in these cells irrespective of the presence or absence of PLP.

• Reyes-Ramirez F, Sawers RG
• Aerobic activation of transcription of the anaerobically inducible Escherichia coli focA-pfl operon by fumarate nitrate regulator.
• FEMS Microbiol Lett. 2006; 255: 262-7
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• Expression of the anaerobically inducible focA-pfl operon in Escherichia coli was activated nearly sevenfold relative to wild-type under aerobic growth conditions by increasing the dosage of the fnr gene on a pBR322-based plasmid (pCH21). No effect on anaerobic expression levels was observed, suggesting that operon expression under these conditions is maximal. Examination of the complex transcript pattern of the focA-pfl operon confirmed that in strains bearing pCH21 all transcripts, with the exception of the promoter 7 transcript, were up-regulated aerobically. Western analysis of strains bearing pCH21 revealed that the fumarate nitrate regulator (FNR) level was increased approximately ninefold relative to the level in strains bearing a single copy of the fnr gene aerobically, but was only overproduced threefold anaerobically. Analysis of an fnr-lacZ fusion indicated that fnr expression was more strongly negatively autoregulated in anaerobic cells compared with aerobic cells when pCH21 was present. Taken together, these findings suggest that high-level overproduction of FNR is prevented anaerobically by active FNR repressing expression of the fnr gene. Furthermore, transcription from promoter 7 of the focA-pfl operon, which depends on both ArcA-P and FNR, cannot be activated aerobically by overproduction of FNR alone, while promoter 6, which is less dependent on ArcA-P, can be activated under these conditions.

• Tse Sum Bui B, Mattioli TA, Florentin D, Bolbach G, Marquet A
• Escherichia coli biotin synthase produces selenobiotin. Further evidence of the involvement of the [2Fe-2S]2+ cluster in the sulfur insertion step.
• Biochemistry. 2006; 45: 3824-34
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• Biotin synthase, a member of the "radical SAM" family, catalyzes the final step of the biotin biosynthetic pathway, namely, the insertion of a sulfur atom into dethiobiotin. The as-isolated enzyme contains a [2Fe-2S](2+) cluster, but the active enzyme requires an additional [4Fe-4S](2+) cluster, which is formed in the presence of Fe(NH(4))(2)(SO(4))(2) and Na(2)S in the in vitro assay. The role of the [4Fe-4S](2+) cluster is to mediate the electron transfer to SAM, while the [2Fe-2S](2+) cluster is involved in the sulfur insertion step. To investigate the selenium version of the reaction, we have depleted the enzyme of its iron and sulfur and reconstituted the resulting apoprotein with FeCl(3) and Na(2)Se to yield a [2Fe-2Se](2+) cluster. This enzyme was assayed in vitro with Na(2)Se in place of Na(2)S to enable the formation of a [4Fe-4Se](2+) cluster. Selenobiotin was produced, but the activity was lower than that of the as-isolated [2Fe-2S](2+) enzyme in the presence of Na(2)S. The [2Fe-2Se](2+) enzyme was additionally assayed with Na(2)S, to reconstitute a [4Fe-4S](2+) cluster, in case the latter was more efficient than a [4Fe-4Se](2+) cluster for the electron transfer. Indeed, the activity was improved, but in that case, a mixture of biotin and selenobiotin was produced. This was unexpected if one considers the [2Fe-2S](2+) center as the sulfur source (either as the ultimate donor or via another intermediate), unless some exchange of the chalcogenide has taken place in the cluster. This latter point was seen in the resonance Raman spectrum of the reacted enzyme which clearly indicated the presence of both the [2Fe-2Se](2+) and [2Fe-2S](2+) clusters. No exchange was observed in the absence of reaction. These observations bring supplementary proof that the [2Fe-2S](2+) cluster is implicated in the sulfur insertion step.

• Jain C
• Overexpression and purification of tagged Escherichia coli proteins using a chromosomal knock-in strategy.
• Protein Expr Purif. 2006; 46: 294-8
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• The purification of recombinant proteins from Escherichia coli (E. coli) has become a standard procedure both for research purposes and in biotechnology. One common way by which this is accomplished is by subcloning the gene of interest into a suitable expression vector and purifying the overexpressed protein using an affinity tag. In some cases, however, subcloning into plasmid vectors can be problematic. An alternative method could be to overexpress the gene of interest from the chromosome. Here, I describe a strategy to juxtapose strong transcriptional and translational sequences in front of any E. coli gene by recombination, which allows the gene product to be expressed in large quantities in the cell, and purified as a tagged protein. An application of this method to create a recombinant strain overexpressing the HrpA protein is described.

• Layer G, Ollagnier-de Choudens S, Sanakis Y, Fontecave M
• Iron-sulfur cluster biosynthesis: characterization of Escherichia coli CYaY as an iron donor for the assembly of [2Fe-2S] clusters in the scaffold IscU.
• J Biol Chem. 2006; 281: 16256-63
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• The biogenesis of iron-sulfur [Fe-S] clusters requires the coordinated delivery of both iron and sulfide. Sulfide is provided by cysteine desulfurases that use L-cysteine as sulfur source. So far, the physiological iron donor has not been clearly identified. CyaY, the bacterial ortholog of frataxin, an iron binding protein thought to be involved in iron-sulfur cluster formation in eukaryotes, is a good candidate because it was shown to bind iron. Nevertheless, no functional in vitro studies showing an involvement of CyaY in [Fe-S] cluster biosynthesis have been reported so far. In this paper we demonstrate for the first time a specific interaction between CyaY and IscS, a cysteine desulfurase participating in iron-sulfur cluster assembly. Analysis of the iron-loaded CyaY protein demonstrated a strong binding of Fe(3+) and a weak binding of Fe(2+) by CyaY. Biochemical analysis showed that the CyaY-Fe(3+) protein corresponds to a mixture of monomer, intermediate forms (dimer-pentamers), and oligomers with the intermediate one corresponding to the only stable and soluble iron-containing form of CyaY. Using spectroscopic methods, this form was further demonstrated to be functional in vitro as an iron donor during [Fe-S] cluster assembly on the scaffold protein IscU in the presence of IscS and cysteine. All of these results point toward a link between CyaY and [Fe-S] cluster biosynthesis, and a possible mechanism for the process is discussed.

• Lehmann C, Begley TP, Ealick SE
• Structure of the Escherichia coli ThiS-ThiF complex, a key component of the sulfur transfer system in thiamin biosynthesis.
• Biochemistry. 2006; 45: 11-9
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• We have determined the crystal structure of the Escherichia coli ThiS-ThiF protein complex at 2.0 A resolution. ThiS and ThiF are bacterial proteins involved in the synthesis of the thiazole moiety of thiamin. ThiF catalyzes the adenylation of the carboxy terminus of ThiS and the subsequent displacement of AMP catalyzed by ThiI-persulfide to give a ThiS-ThiI acyl disulfide. Disulfide interchange, involving Cys184 on ThiF, then generates the ThiS-ThiF acyl disulfide, which functions as the sulfur donor for thiazole formation. ThiS is a small 7.2 kDa protein that structurally resembles ubiquitin and the molybdopterin biosynthetic protein MoaD. ThiF is a 27 kDa protein with distinct sequence and structural similarity to the ubiquitin activating enzyme E1 and the molybdopterin biosynthetic protein MoeB. The ThiF-ThiS structure clarifies the mechanism of the sulfur transfer chemistry involved in thiazole biosynthesis.

• Sperandeo P, Pozzi C, Deho G, Polissi A
• Non-essential KDO biosynthesis and new essential cell envelope biogenesis genes in the Escherichia coli yrbG-yhbG locus.
• Res Microbiol. 2006; 157: 547-58
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• In Escherichia coli and most Gram-negative bacteria, KDO (3-deoxy-D-manno-octulosonate), a component of the lipopolysaccharide inner core, is essential for outer membrane biogenesis and cell viability. Two recently identified genes involved in KDO biosynthesis, kdsD and kdsC, belong to the yrbG-yhbG locus where four additional ORFs (yrbG, yrbK, yhbN and yhbG) with unknown function are located. We have constructed six conditional expression mutants in which the arabinose-inducible araBp promoter is respectively located upstream of each gene of the locus. Complementation analysis of these mutants indicates that the locus is organized in at least three operons and that the three distal genes (yrbK, yhbN and yhbG) are essential for E. coli viability. Surprisingly, kdsD and kdsC (encoding a D-arabinose 5-phosphate isomerase and a KDO 8-phosphate phosphatase, respectively) were shown to be non-essential, indicating genetic redundancy for these two functions. A preliminary characterization of the arabinose-dependent mutants under permissive conditions and upon depletion revealed increased sensitivity to hydrophobic toxic chemicals, suggesting that the mutants have a defective outer membrane. These genes may thus be implicated in cell envelope integrity.

• Mahren S, Schnell H, Braun V
• Occurrence and regulation of the ferric citrate transport system in Escherichia coli B, Klebsiella pneumoniae, Enterobacter aerogenes, and Photorhabdus luminescens.
• Arch Microbiol. 2005; 184: 175-86
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• In Escherichia coli K-12, transcription of the ferric citrate transport genes fecABCDE is initiated by binding of diferric dicitrate to the outer membrane protein FecA which elicits a signaling cascade from the cell surface to the cytoplasm. The FecI sigma factor is only active in the presence of FecR, which transfers the signal across the cytoplasmic membrane. In other bacteria, fecIRA homologues control iron transport gene transcription by siderophores other than citrate. However, in most cases, the FecI homologues are active in the absence of the FecR homologues, which might function as anti-sigma factors. Since not all E. coli strains contain a fec system, we determined the occurrence of fec genes in selected Enterobacteriaceae and the dependence of FecI activity on FecR. Incomplete FecIRA systems were chromosomally encoded in Enterobacter aerogenes strains and plasmid-encoded in K. pneumoniae. E. coli B, Photorhabdus luminescens and one of three Klebsiella pneumoniae strains had a functional FecIRA regulatory system as in E. coli K-12. The cytoplasmic N-terminal FecR fragments caused constitutive FecI activity in the absence of ferric citrate. The PCR-generated mutant FecI(D40G) was inactive and FecI(S15P) was partially active. FecR of E. coli K-12 activated FecI of all tested strains except FecI encoded on the virulence plasmid pLVPK of K. pneumoniae, which differed from E. coli K-12 FecI by having mutations in region 4, which is important for interaction with FecR. The C-terminally truncated FecR homologue of pLVPK was inactive. pLVPK-encoded FecA contains a 38-residue sequence in front of the signal sequence that did not prevent processing and proper integration of FecA into the outer membrane of E. coli and lacks the signaling sequence required for transcription initiation of the fec transport genes, making it induction-incompetent but transport-competent. The evidence indicates that fecIRABCDE genes are acquired by horizontal DNA transfer and can undergo debilitating mutations.

• Beckett D
• The Escherichia coli biotin regulatory system: a transcriptional switch.
• J Nutr Biochem. 2005; 16: 411-5
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• The ability of any organism to survive depends, in part, on mechanisms that enable it to modify its patterns of gene expression in response to extra- and intracellular signals. In the classical response mechanisms, a small molecule signal impinges on either an extra- or intracellular receptor, and through a series of events the signal is ultimately transmitted to transcription regulatory proteins. An alternative to this classical mechanism is provided by multi-functional transcription factors. These proteins function directly in transcription as well as in at least one additional cellular process. An example of this class of proteins includes the dimerization cofactor of hepatocyte nuclear factor (DcoH), which serves as an enzyme involved in regeneration of the tetra-hydrobiopterin cofactor and as a factor that stabilizes the dimerization of the hepatocyte nuclear transcription factor (Mendel DB, Khavari PA, Conley PB, Graves MK, Hansen LP, Admon A, et al. Characterization of a cofactor that regulates dimerization of a mammalian homeodomain protein. Science 1991;254:1762-7; Citron BA, Davis MD, Milstien S, Gutierrez J, Mendel DB, Crabtree GR. Identity of 4a-carbinolamine dehydratase, a component of the phenylalanine hydroxylation system, and DCoH, a transregulator of homeodomain proteins. Proc Natl Acad Sci U S A 1992;89:11891-4). Another example is the protein PutA, a redox enzyme involved in proline utilization and a regulator of transcription of the genes involved in proline utilization (Ostrovsky de Spicer P, Maloy S. Puta protein, a membrane-associated flavin dehydrogenase, acts as a redox-dependent transcriptional regulator. Proc Natl Acad Sci U S A 1993;90:4295-8). While several proteins of this class have been identified, their mechanisms of functional switching remain to be elucidated.

• Gurung B, Yu L, Xia D, Yu CA
• The iron-sulfur cluster of the Rieske iron-sulfur protein functions as a proton-exiting gate in the cytochrome bc(1) complex.
• J Biol Chem. 2005; 280: 24895-902
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• The destruction of the Rieske iron-sulfur cluster ([2Fe-2S]) in the bc(1) complex by hematoporphyrin-promoted photoinactivation resulted in the complex becoming proton-permeable. To study further the role of this [2Fe-2S] cluster in proton translocation of the bc(1) complex, Rhodobacter sphaeroides mutants expressing His-tagged cytochrome bc(1) complexes with mutations at the histidine ligands of the [2Fe-2S] cluster were generated and characterized. These mutants lacked the [2Fe-2S] cluster and possessed no bc(1) activity. When the mutant complex was co-inlaid in phospholipid vesicles with intact bovine mitochondrial bc(1) complex or cytochrome c oxidase, the proton ejection, normally observed in intact reductase or oxidase vesicles during the oxidation of their corresponding substrates, disappeared. This indicated the creation of a proton-leaking channel in the mutant complex, whose [2Fe-2S] cluster was lacking. Insertion of the bc(1) complex lacking the head domain of the Rieske iron-sulfur protein, removed by thermolysin digestion, into PL vesicles together with mitochondrial bc(1) complex also rendered the vesicles proton-permeable. Addition of the excess purified head domain of the Rieske iron-sulfur protein partially restored the proton-pumping activity. These results indicated that elimination of the [2Fe-2S] cluster in mutant bc(1) complexes opened up an otherwise closed proton channel within the bc(1) complex. It was speculated that in the normal catalytic cycle of the bc(1) complex, the [2Fe-2S] cluster may function as a proton-exiting gate.

• Heidenreich T, Wollers S, Mendel RR, Bittner F
• Characterization of the NifS-like domain of ABA3 from Arabidopsis thaliana provides insight into the mechanism of molybdenum cofactor sulfuration.
• J Biol Chem. 2005; 280: 4213-8
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• The molybdenum cofactor sulfurase ABA3 from Arabidopsis thaliana specifically regulates the activity of the molybdenum enzymes aldehyde oxidase and xanthine dehydrogenase by converting their molybdenum cofactor from the desulfo-form into the sulfo-form. ABA3 is a two-domain protein with an NH2-terminal domain sharing significant similarities to NifS proteins that catalyze the decomposition of l-cysteine to l-alanine and elemental sulfur for iron-sulfur cluster synthesis. Although different in its physiological function, the mechanism of ABA3 for sulfur mobilization was found to be similar to NifS proteins. The protein binds a pyridoxal phosphate cofactor and a substrate-derived persulfide intermediate, and site-directed mutagenesis of strictly conserved binding sites for the cofactor and the persulfide demonstrated that they are essential for molybdenum cofactor sulfurase activity. In vitro, the NifS-like domain of ABA3 activates aldehyde oxidase and xanthine dehydrogenase in the absence of the C-terminal domain, but in vivo, the C-terminal domain is required for proper activation of both target enzymes. In addition to its cysteine desulfurase activity, ABA3-NifS also exhibits selenocysteine lyase activity. Although l-selenocysteine is unlikely to be a natural substrate for ABA3, it is decomposed more efficiently than l-cysteine. Besides mitochondrial AtNFS1 and plastidial AtNFS2, which are both proposed to be involved in iron-sulfur cluster formation, ABA3 is proposed to be a third and cytosolic NifS-like cysteine desulfurase in A. thaliana. However, the sulfur transferase activity of ABA3 is used for post-translational activation of molybdenum enzymes rather than for iron-sulfur cluster assembly.

• Choi-Rhee E, Cronan JE
• Biotin synthase is catalytic in vivo, but catalysis engenders destruction of the protein.
• Chem Biol. 2005; 12: 461-8
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• Biotin synthase is responsible for the synthesis of biotin from dethiobiotin and sulfur. Although the name of the protein implies that it functions as an enzyme, it has been consistently reported that biotin synthase produces <1 molecule of biotin per molecule of protein in vitro. Moreover, the source of the biotin sulfur atom has been reported to be the [2Fe-2S] center of the protein. Biotin synthase has therefore been designated as a substrate or reactant rather than an enzyme. We report in vivo experiments demonstrating that biotin synthase is catalytic but that catalysis puts the protein at risk of proteolytic destruction.

• Beeler JA, Tang WJ
• Expression and purification of soluble adenylyl cyclase from Escherichia coli.
• Methods Mol Biol. 2004; 237: 39-53
• Display abstract

• This chapter outlines methods to purify soluble adenylyl cyclase (AC7) expressed in an Escherichia coli (E. coli) heterologous expression system. Guidelines are provided for constructing the expression plasmids, optimizing expression, culturing, and purifying the proteins. Purification requires two chromatographic steps. A histidine tag (H6) is incorporated into the expression vector and utilized for affinity purification on a Ni-NTA column. Subsequently, an anion exchange column is employed to further purify the protein.

• Hanzelmann P et al.
• Characterization of MOCS1A, an oxygen-sensitive iron-sulfur protein involved in human molybdenum cofactor biosynthesis.
• J Biol Chem. 2004; 279: 34721-32
• Display abstract

• The human proteins MOCS1A and MOCS1B catalyze the conversion of a guanosine derivative to precursor Z during molybdenum cofactor biosynthesis. MOCS1A shares homology with S-adenosylmethionine (AdoMet)-dependent radical enzymes, which catalyze the formation of protein and/or substrate radicals by reductive cleavage of AdoMet through a [4Fe-4S] cluster. Sequence analysis of MOCS1A showed two highly conserved cysteine motifs, one near the N terminus and one near the C terminus. MOCS1A was heterologously expressed in Escherichia coli and purified under aerobic and anaerobic conditions. Individual mutations of the conserved cysteines to serine revealed that all are essential for synthesis of precursor Z in vivo. The type and properties of the iron-sulfur (FeS) clusters were investigated using a combination of UV-visible absorption, variable temperature magnetic circular dichroism, resonance Raman, Mossbauer, and EPR spectroscopies coupled with iron and acid-labile sulfide analyses. The results indicated that anaerobically purified MOCS1A is a monomeric protein containing two oxygen-sensitive FeS clusters, each coordinated by only three cysteine residues. A redox-active [4Fe-4S](2+,+) cluster is ligated by an N-terminal CX(3)CX(2)C motif as is the case with all other AdoMet-dependent radical enzymes investigated thus far. A C-terminal CX(2)CX(13)C motif that is unique to MOCS1A and its orthologs primarily ligates a [3Fe-4S](0) cluster. However, MOCS1A could be reconstituted in vitro under anaerobic conditions to yield a form containing two [4Fe-4S](2+) clusters. The N-terminal [4Fe-4S](2+) cluster was rapidly degraded by oxygen via a semistable [2Fe-2S](2+) cluster intermediate, and the C-terminal [4Fe-4S](2+) cluster was rapidly degraded by oxygen to yield a semistable [3Fe-4S](0) cluster intermediate.

• Dorrestein PC, Zhai H, McLafferty FW, Begley TP
• The biosynthesis of the thiazole phosphate moiety of thiamin: the sulfur transfer mediated by the sulfur carrier protein ThiS.
• Chem Biol. 2004; 11: 1373-81
• Display abstract

• Thiamin-pyrophosphate is an essential cofactor in all living systems. The biosynthesis of both the thiazole and the pyrimidine moieties of this cofactor involves new biosynthetic chemistry. Thiazole-phosphate synthase (ThiG) catalyses the formation of the thiazole moiety of thiamin-pyrophosphate from 1-deoxy-D-xylulose-5-phosphate (DXP), dehydroglycine and the sulfur carrier protein (ThiS), modified on its carboxy terminus as a thiocarboxylate (ThiS-thiocarboxylate). Thiazole biosynthesis is initiated by the formation of a ThiG/DXP imine, which then tautomerizes to an amino-ketone. In this paper we study the sulfur transfer from ThiS-thiocarboxylate to this amino-ketone and trap a new thioenolate intermediate. Surprisingly, thiazole formation results in the replacement of the ThiS-thiocarboxylate sulfur with an oxygen from DXP and not from the buffer, as shown by electrospray ionization Fourier transform mass spectrometry (ESI-FTMS) using (18)O labeling of the 13C-, 15N-depleted protein. These observations further clarify the mechanism of the complex thiazole biosynthesis in bacteria.

• Ashraf SS, Benson RE, Payne ES, Halbleib CM, Gron H
• A novel multi-affinity tag system to produce high levels of soluble and biotinylated proteins in Escherichia coli.
• Protein Expr Purif. 2004; 33: 238-45
• Display abstract

• We describe here a novel multi-affinity tag vector that can be used to produce high levels of soluble, in vivo biotinylated proteins in Escherichia coli. This system combines the solubility-enhancing ability of maltose-binding protein (MBP), the versatility of the hexahistidine tag (His(6)), and the site-specific in vivo biotinylation of a 15-amino acid tag (AviTag). We used this multi-tag system in an attempt to improve expression levels of two prokaryotic proteins-elongation factor Tu (TufB) and DNA gyrase subunit A (GyrA)-as well as two eukaryotic nuclear receptors-glucocorticoid receptor (GR) and small heterodimer partner (SHP). The multi-tag system not only vastly improved the expression of the two prokaryotic proteins tested, but also yielded complete, site-specific, in vivo biotinylation of these proteins. The results obtained from the TufB expression and purification are presented and discussed in detail. The nuclear receptors, though soluble as fusion partners, failed to remain soluble once the MBP tag was cleaved. Despite this limitation of the system, the multi-affinity tag approach is a useful system that can improve expression of some otherwise insoluble or poorly expressing proteins, to obtain homogeneous, purified, fully biotinylated protein for downstream applications.

• Mishina Y, Chen LX, He C
• Preparation and characterization of the native iron(II)-containing DNA repair AlkB protein directly from Escherichia coli.
• J Am Chem Soc. 2004; 126: 16930-6
• Display abstract

• The Escherichia coli AlkB protein was recently found to repair cytotoxic DNA lesions 1-methyladenine and 3-methylcytosine by using a novel iron-catalyzed oxidative demethylation mechanism. This protein belongs to a family of 2-ketoglutarate-Fe(II)-dependent dioxygenase proteins that utilize iron and 2-ketoglutarate to activate dioxygen for oxidation reactions. We report here the overexpression and isolation of the native Fe(II)-AlkB with a bound cofactor, 2-ketoglutarate, directly from E. coli. UV-vis measurements showed an absorption peak at 560 nm, which is characteristic of a bidentate 2-ketoglutarate bound to an iron(II) ion. Addition of excess amounts of single-stranded DNA to this isolated Fe(II)-AlkB protein caused a 9 nm shift of the 560 nm band to a higher energy, indicating a DNA-binding-induced geometry change of the active site. X-ray absorption spectra of the active site iron(II) in AlkB suggest a five-coordinate iron(II) center in the protein itself and a centrosymmetric six-coordinate iron(II) site upon addition of single-stranded DNA. This geometry change may play important roles in the DNA damage-searching and damage-repair functions of AlkB. These results provide direct evidence for DNA binding to AlkB which modulates the active site iron(II) geometry. The isolation of the native Fe(II)-AlkB also allows for further investigation of the iron(II) center and detailed mechanistic studies of the dioxygen-activation and damage-repair reactions performed by AlkB.

• Cosper MM, Jameson GN, Hernandez HL, Krebs C, Huynh BH, Johnson MK
• Characterization of the cofactor composition of Escherichia coli biotin synthase.
• Biochemistry. 2004; 43: 2007-21
• Display abstract

• The cofactor content of in vivo, as-isolated, and reconstituted forms of recombinant Escherichia coli biotin synthase (BioB) has been investigated using the combination of UV-visible absorption, resonance Raman, and Mossbauer spectroscopies along with parallel analytical and activity assays. In contrast to the recent report that E. coli BioB is a pyridoxal phosphate (PLP)-dependent enzyme with intrinsic cysteine desulfurase activity (Ollagnier-deChoudens, S., Mulliez, E., Hewitson, K. S., and Fontecave, M. (2002) Biochemistry 41, 9145-9152), no evidence for PLP binding or for PLP-induced cysteine desulfurase or biotin synthase activity was observed with any of the forms of BioB investigated in this work. The results confirm that BioB contains two distinct Fe-S cluster binding sites. One site accommodates a [2Fe-2S](2+) cluster with partial noncysteinyl ligation that can only be reconstituted in vitro in the presence of O(2). The other site accommodates a [4Fe-4S](2+,+) cluster that binds S-adenosylmethionine (SAM) at a unique Fe site of the [4Fe-4S](2+) cluster and undergoes O(2)-induced degradation via a distinct type of [2Fe-2S](2+) cluster intermediate. In vivo Mossbauer studies show that recombinant BioB in anaerobically grown cells is expressed exclusively in an inactive form containing only the as-isolated [2Fe-2S](2+) cluster and demonstrate that the [2Fe-2S](2+) cluster is not a consequence of overexpressing the recombinant enzyme under aerobic growth conditions. Overall the results clarify the confusion in the literature concerning the Fe-S cluster composition and the in vitro reconstitution and O(2)-induced cluster transformations that are possible for recombinant BioB. In addition, they provide a firm foundation for assessing cluster transformations that occur during turnover and the catalytic competence of the [2Fe-2S](2+) cluster as the immediate S-donor for biotin biosynthesis.

• Smith MN, Crane RA, Keates RA, Wood JM
• Overexpression, purification, and characterization of ProQ, a posttranslational regulator for osmoregulatory transporter ProP of Escherichia coli.
• Biochemistry. 2004; 43: 12979-89
• Display abstract

• ProP is an osmosensor and osmoregulatory transporter in Escherichia coli. Osmotic activation of ProP is attenuated 5-fold in the absence of soluble protein ProQ, but proQ lesions do not influence proP transcription or ProP levels. The mechanism by which ProQ amplifies ProP activity is unknown. Putative proQ orthologues are found in Gram-negative bacteria (only), but none have known functions. ProQ was overexpressed to low and high levels with and without a C-terminal histidine tag (His(6)). Plasmid-encoded ProQ or ProQ-His(6) complemented in-frame chromosomal deletion DeltaproQ676, restoring ProP activity. After overexpression, both proteins were poorly soluble unless cells were lysed in media of high salinity. ProQ copurified with DNA binding proteins of similar size (HU and a histone-like protein) by ion exchange and exclusion chromatographies, whereas ProQ-His(6) could be purified to homogeneity by nickel chelate affinity chromatography. Sequence-based analysis and modeling suggest that ProQ includes distinct N- and C-terminal domains linked by an unstructured sequence. The N-terminal domain can be modeled on the crystal structure of alpha-helical RNA binding protein FinO, whereas the C-terminal domain can be modeled on an SH3-like domain (beta-structure). Both ProQ and ProQ-His(6) appeared to be monomeric, though the higher Stokes radius of ProQ-His(6) may reflect altered domain interactions. The measured secondary structure content of ProQ (circular dichroism (CD) spectroscopy) contrasted with sequence-based prediction but was as expected if the spectrum of the C-terminal domain is analogous to those reported for SH3 domains. The CD spectrum of ProQ was pH- but not NaCl-sensitive.

• Grawert T et al.
• IspH protein of Escherichia coli: studies on iron-sulfur cluster implementation and catalysis.
• J Am Chem Soc. 2004; 126: 12847-55
• Display abstract

• The ispH gene of Escherichia coli specifies an enzyme catalyzing the conversion of 1-hydroxy-2-methyl-2-(E)-butenyl diphosphate into a mixture of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) in the nonmevalonate isoprenoid biosynthesis pathway. The implementation of a gene cassette directing the overexpression of the isc operon involved in the assembly of iron-sulfur clusters into an Escherichia coli strain engineered for ispH gene expression increased the catalytic activity of IspH protein anaerobically purified from this strain by a factor of at least 200. For maximum catalytic activity, flavodoxin and flavodoxin reductase were required in molar concentrations of 40 and 12 microM, respectively. EPR experiments as well as optical absorbance indicate the presence of a [3Fe-4S](+) cluster in IspH protein. Among 4 cysteines in total, the 36 kDa protein carries 3 absolutely conserved cysteine residues at the amino acid positions 12, 96, and 197. Replacement of any of the conserved cysteine residues reduced the catalytic activity by a factor of more than 70 000.

• Kriek M, Peters L, Takahashi Y, Roach PL
• Effect of iron-sulfur cluster assembly proteins on the expression of Escherichia coli lipoic acid synthase.
• Protein Expr Purif. 2003; 28: 241-5
• Display abstract

• Lipoic Acid Synthase (LipA) can accommodate a [4Fe-4S] cluster that is thought to be essential for the insertion of sulfur into an octanoyl substrate during the biosynthesis of lipoic acid. With the objective of improving soluble holo-LipA expression, a series of multi-cistronic plasmids were constructed carrying lipA in combination with one of the three systems: groE/SL, trxA, or the isc operon. Co-expression of lipA with the isc operon approximately trebled the isolated yield of soluble LipA and resulted in efficient assembly of the Fe-S cluster. This strategy may be helpful in the soluble expression of a wide range of Fe-S cluster-dependent proteins.

• Gubernator B, Seidler A, Rogner M, Szczepaniak A
• Overexpression and reconstitution of a Rieske iron-sulfur protein from the higher plant.
• Protein Expr Purif. 2003; 29: 8-14
• Display abstract

• The iron-sulfur protein subunit, known as the Rieske protein, is one of the central components of the cytochrome b(6)f complex residing in chloroplast and cyanobacterial thylakoid membranes. We have constructed plasmids for overexpression in Escherichia coli of full-length and truncated Rieske (PetC) proteins from the Spinacia oleracea fused to MalE. Overexpressed fusion proteins were predominantly found (from 55 to 70%) in cytoplasm in a soluble form. The single affinity chromatography step (amylose resine) was used to purify about 15mg of protein from 1 liter of E. coli culture. The isolated proteins were electrophoretically pure and could be used for further experiments. The NifS-like protein IscS from the cyanobacterium Synechocystis PCC 6803 mediates the incorporation of 2Fe-2S clusters into apoferredoxin and cyanobacterial Rieske apoprotein in vitro. Here, we used the recombinant IscS protein for the enzymatic reconstitution of the iron-sulfur cluster into full-length Rieske fusion and truncated Rieske fused proteins. Characterization by EPR spectroscopy of the reconstituted proteins demonstrated the presence of a 2Fe-2S cluster in both full-length and truncated Rieske fusion proteins.

• Kiyasu T, Asakura A, Nagahashi Y, Hoshino T
• Biotin synthase of Bacillus subtilis shows less reactivity than that of Escherichia coli in in vitro reaction systems.
• Arch Microbiol. 2002; 179: 26-32
• Display abstract

• The biotin synthases of Bacillus subtilis and Escherichia coli were compared in a physiological reduction system using cell-free extracts and in a artificial reduction system using photo-reduced deazariboflavin. The biotin synthase of B. subtilis was less active than that of E. coli in both reaction systems and showed at least ten-fold less biotin-forming activity than that of E. coli in the artificial reduction system. The physiological reduction system using the biotin synthases and cell-free extracts of B. subtilis and E. coli showed species specificity. The results suggest that the activity of the physiological reduction system of B. subtilisis weaker than that of E. coli. Addition of excess dethiobiotin inhibited biotin formation by growing cells of B. subtilis, but not by E. coli.

• Farh L, Hwang SY, Steinrauf L, Chiang HJ, Shiuan D
• Structure-function studies of Escherichia coli biotin synthase via a chemical modification and site-directed mutagenesis approach.
• J Biochem. 2001; 130: 627-35
• Display abstract

• In Escherichia coli, biotin synthase (bioB gene product) catalyzes the key step in the biotin biosynthetic pathway, converting dethiobiotin (DTB) to biotin. Previous studies have demonstrated that BioB is a homodimer and that each monomer contains an iron-sulfur cluster. The purified BioB protein, however, does not catalyze the formation of biotin in a conventional fashion. The sulfur atom in the iron-sulfur cluster or from the cysteine residues in BioB have been suggested to act as the sulfur donor to form the biotin molecule, and yet unidentified factors were also proposed to be required to regenerate the active enzyme. In order to understand the catalytic mechanism of BioB, we employed an approach involving chemical modification and site-directed mutagenesis. The properties of the modified and mutated BioB species were examined, including DTB binding capability, biotin converting activity, and Fe(2+) content. From our studies, four cysteine residues (Cys 53, 57, 60, and 97) were assigned as the ligands of the iron-sulfur cluster, and Cys to Ala mutations completely abolished biotin formation activity. Two other cysteine residues (Cys 128 and 188) were found to be involved mainly in DTB binding. The tryptophan and histidine residues were suggested to be involved in DTB binding and dimer formation, respectively. The present study also reveals that the iron-sulfur cluster with its ligands are the key components in the formation of the DTB binding site. Based on the current results, a refined model for the reaction mechanism of biotin synthase is proposed.

• Marquet A
• Enzymology of carbon-sulfur bond formation.
• Curr Opin Chem Biol. 2001; 5: 541-9
• Display abstract

• Mobilization of the sulfur of cysteine as persulfide is the first step of sulfur transfer into thiamin, molydopterin, 4-thiouridine, biotin and lipoic acid, but then the pathways diverge completely. For the first three compounds, one or several proteinic persulfides are involved, ending in the nucleophilic attack of a sulfur, persulfide, sulfide or thiocarboxylate on a carbonyl equivalent. Several proteins have been newly characterized, revealing homologies between the three biosynthetic routes and evolutionary relationships. In the case of biotin, and very probably of lipoic acid, the sulfur is transferred as sulfide into the [Fe-S] center of the enzyme. This [Fe-S] center is the ultimate sulfur donor, which quenches a carbon radical on the substrate. This radical is produced by homolytic cleavage of a C-H bond by a deoxyadenosyl radical arising from the reduction of S-adenosylmethionine.

• Leimkuhler S, Rajagopalan KV
• A sulfurtransferase is required in the transfer of cysteine sulfur in the in vitro synthesis of molybdopterin from precursor Z in Escherichia coli.
• J Biol Chem. 2001; 276: 22024-31
• Display abstract

• It has been shown that conversion of precursor Z to molybdopterin (MPT) by Escherichia coli MPT synthase entails the transfer of the sulfur atom of the C-terminal thiocarboxylate from the small subunit of the synthase to generate the dithiolene group of MPT and that the moeB mutant of E. coli contains inactive MPT synthase devoid of the thiocarboxylate. The data presented here demonstrate that l-cysteine can serve as the source of the sulfur for the biosynthesis of MPT in vitro but only in the presence of a persulfide-containing sulfurtransferase such as IscS, cysteine sulfinate desulfinase (CSD), or CsdB. A fully defined in vitro system has been developed in which an inactive form of MPT synthase can be activated by incubation with MoeB, Mg-ATP, l-cysteine, and one of the NifS-like sulfurtransferases, and the addition of precursor Z to the in vitro system gives rise to MPT formation. The use of radiolabeled l-[(35)S]cysteine has demonstrated that both sulfurs of the dithiolene group of MPT originate from l-cysteine. It was found that MPT can be produced from precursor Z in an E. coli iscS mutant strain, indicating that IscS is not required for the in vivo sulfuration of MPT synthase. A comparison of the ability of the three sulfurtransferases to provide the sulfur for MPT formation showed the highest activity for CSD in the in vitro system.

• Gutzke G, Fischer B, Mendel RR, Schwarz G
• Thiocarboxylation of molybdopterin synthase provides evidence for the mechanism of dithiolene formation in metal-binding pterins.
• J Biol Chem. 2001; 276: 36268-74
• Display abstract

• Molybdopterin (MPT) is a pyranopterin with a unique dithiolene group coordinating molybdenum (Mo) or tungsten (W) in all Mo- and W-enzymes except nitrogenase. In Escherichia coli, MPT is formed by incorporation of two sulfur atoms into precursor Z, which is catalyzed by MPT synthase. The recently solved crystal structure of MPT synthase (Rudolph, M. J., Wuebbens, M. M., Rajagopalan, K. V., and Schindelin, H. (2000) Nat. Struct. Biol. 8, 42-46) shows the heterotetrameric nature of the enzyme that is composed of two small (MoaD) and two large subunits (MoaE). According to sequence and structural similarities among MoaD, ubiquitin, and ThiS, a thiocarboxylation of the C terminus of MoaD is proposed that would serve as the source of sulfur that is transferred to precursor Z. Here, we describe the in vitro generation of carboxylated and thiocarboxylated MoaD. Both forms of MoaD are monomeric and are able to form a heterotetrameric complex after coincubation in equimolar ratios with MoaE. Only the thiocarboxylated MPT synthase complex was found to be able to convert precursor Z in vitro to MPT. Slight but significant differences between the carboxylated and the thiocarboxylated MPT synthase can be seen using size exclusion chromatography. A two-step reaction of MPT synthesis is proposed where the dithiolene is generated by two thiocarboxylates derived from a single tetrameric MPT synthase.

• Hewitson KS, Baldwin JE, Shaw NM, Roach PL
• Mutagenesis of the proposed iron-sulfur cluster binding ligands in Escherichia coli biotin synthase.
• FEBS Lett. 2000; 466: 372-6
• Display abstract

• Biotin synthase (BioB) is a member of a family of enzymes that includes anaerobic ribonucleotide reductase and pyruvate formate lyase activating enzyme. These enzymes all use S-adenosylmethionine during turnover and contain three highly conserved cysteine residues that may act as ligands to an iron-sulfur cluster required for activity. Three mutant enzymes of BioB have been made, each with one cysteine residue (C53, 57, 60) mutated to alanine. All three mutant enzymes were inactive, but they still exhibited the characteristic UV-visible spectrum of a [2Fe-2S]2+ cluster similar to that of the wild-type enzyme.

• Bui BT, Escalettes F, Chottard G, Florentin D, Marquet A
• Enzyme-mediated sulfide production for the reconstitution of [2Fe-2S] clusters into apo-biotin synthase of Escherichia coli. Sulfide transfer from cysteine to biotin.
• Eur J Biochem. 2000; 267: 2688-94
• Display abstract

• We previously showed that biotin synthase in which the (Fe-S) cluster was labelled with 34S by reconstitution donates 34S to biotin [B. Tse Sum Bui, D. Florentin, F. Fournier, O. Ploux, A. Mejean & A. Marquet (1998) FEBS Lett. 440, 226-230]. We therefore proposed that the source of sulfur was very likely the (Fe-S) centre. This depletion of sulfur from the cluster during enzymatic reaction could explain the absence of turnover of the enzyme which means that to restore a catalytic activity, the clusters have to be regenerated. In this report, we show that the NifS protein from Azotobacter vinelandii and C-DES from Synechocystis as well as rhodanese from bovine liver can mobilize the sulfur, respectively, from cysteine and thiosulfate for the formation of a [2Fe-2S] cluster in the apoprotein of Escherichia coli biotin synthase. The reconstituted enzymes were as active as the native enzyme. When [35S]cysteine was used during the reconstitution experiments in the presence of NifS, labelled (Fe35S) biotin synthase was obtained. This enzyme produced [35S]biotin, confirming the results obtained with the 34S-reconstituted enzyme. NifS was also effective in mobilizing selenium from selenocystine to produce an (Fe-Se) cluster. However, though NifS could efficiently reconstitute holobiotin synthase from the apoform, starting from cysteine, these two effectors had no significant effect on the turnover of the enzyme in the in vitro assay.

• McIver L, Baxter RL, Campopiano DJ
• Identification of the [Fe-S] cluster-binding residues of Escherichia coli biotin synthase.
• J Biol Chem. 2000; 275: 13888-94
• Display abstract

• The gene encoding Escherichia coli biotin synthase (bioB) has been expressed as a histidine fusion protein, and the protein was purified in a single step using immobilized metal affinity chromatography. The His(6)-tagged protein was fully functional in in vitro and in vivo biotin production assays. Analysis of all the published bioB sequences identified a number of conserved residues. Single point mutations, to either serine or threonine, were carried out on the four conserved (Cys-53, Cys-57, Cys-60, and Cys-188) and one non-conserved (Cys-288) cysteine residues, and the purified mutant proteins were tested both for ability to reconstitute the [2Fe-2S] clusters of the native (oxidized) dimer and enzymatic activity. The C188S mutant was insoluble. The wild-type and four of the mutant proteins were characterized by UV-visible spectroscopy, metal and sulfide analysis, and both in vitro and in vivo biotin production assays. The molecular masses of all proteins were verified using electrospray mass spectrometry. The results indicate that the His(6) tag and the C288T mutation have no effect on the activity of biotin synthase when compared with the wild-type protein. The C53S, C57S, and C60S mutant proteins, both as prepared and reconstituted, were unable to covert dethiobiotin to biotin in vitro and in vivo. We conclude that three of the conserved cysteine residues (Cys-53, Cys-57, and Cys-60), all of which lie in the highly conserved "cysteine box" motif, are crucial for [Fe-S] cluster binding, whereas Cys-188 plays a hitherto unknown structural role in biotin synthase.

• Begley TP, Xi J, Kinsland C, Taylor S, McLafferty F
• The enzymology of sulfur activation during thiamin and biotin biosynthesis.
• Curr Opin Chem Biol. 1999; 3: 623-9
• Display abstract

• The thiamin and biotin biosynthetic pathways utilize elaborate strategies for the transfer of sulfur from cysteine to cofactor precursors. For thiamin, the sulfur atom of cysteine is transferred to a 66-amino-acid peptide (ThiS) to form a carboxy-terminal thiocarboxylate group. This sulfur transfer requires three enzymes and proceeds via a ThiS-acyladenylate intermediate. The biotin synthase Fe-S cluster functions as the immediate sulfur donor for biotin formation. C-S bond formation proceeds via radical intermediates that are generated by hydrogen atom transfer from dethiobiotin to the adenosyl radical. This radical is formed by the reductive cleavage of S-adenosylmethionine by the reduced Fe-S cluster of biotin synthase.

• Bui BT, Florentin D, Fournier F, Ploux O, Mejean A, Marquet A
• Biotin synthase mechanism: on the origin of sulphur.
• FEBS Lett. 1998; 440: 226-30
• Display abstract

• Biotin synthase catalyses the last step of the biosynthesis of biotin in microorganisms and plants. The active protein isolated from Bacillus sphaericus and Escherichia coli contains an iron-sulphur (FeS) cluster. The native enzymes were depleted of their iron and inorganic sulphide and the resulting apoenzymes were chemically reconstituted with FeCl3 and Na2[34S] to give labelled (Fe34S) enzymes. These enzymes were functional and when assayed in vitro produced labelled biotin containing about 65% of 34S. These data strongly support the hypothesis that the sulphur of biotin is derived from the (FeS) centre of the enzyme.

• Bui BT, Marquet A
• Biotin synthase of Bacillus sphaericus.
• Methods Enzymol. 1997; 279: 356-62

• Salib AG, Frappier F, Guillerm G, Marquet A
• On the mechanism of conversion of dethiobiotin to biotin in Escherichia coli. III. Isolation of an intermediate in the biosynthesis of biotin from dethiobiotin.
• Biochem Biophys Res Commun. 1979; 88: 312-9

• White RH
• 4-Hydroxybenzyl alcohol. A metabolite produced during the biosynthesis of thiamine in Escherichia coli.
• Biochim Biophys Acta. 1979; 583: 55-62
• Display abstract

• 4-Hydroxybenzyl alcohol was identified by gas chromatography-mass spectrometry as a metabolite of Escherichia coli when it is grown on a medium containing no thiamine or 4-methyl-5-beta-hydroxyethyl thiazole. 4-Hydroxybenzyl alcohol was found to be derived from L-tyrosine and the amount produced was found to be inhibited by the addition of thiamine to the growth medium. The amount of 4-hydroxybenzyl alcohol produced, as measured by isotopic dilution, was shown to be equivalent to the amount of thiamine formed. Based on these observations, it was concluded that 4-hydroxybenzyl alcohol is the cleavage product produced during the biosynthesis of the thiazole moiety of thiamine from tyrosine.

• Rudiger H
• A new activator in the vitamin b(12)-dependent methionine biosynthesis of Escherichia coli.
• FEBS Lett. 1972; 27: 39-40

• Iwashima A, Takahashi K, Nose Y
• Overproduction of hydroxymethylpyrimidine by a thiamine regulatory mutant of Escherichia coli.
• J Vitaminol (Kyoto). 1971; 17: 43-8

• Rudiger H, Jaenicke L
• [A regulating factor of methionine biosynthesis].
• Naturwissenschaften. 1970; 57: 132-132

• Shimada K, Nagase Y, Matsumoto U
• Improved selective tritiation of dethiobiotin by reductive desulfurization from biotin.
• Chem Pharm Bull (Tokyo). 1968; 16: 1632-4

• Eakin RE, Eakin EA
• A BIOSYNTHESIS OF BIOTIN.
• Science. 1942; 96: 187-8

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