Biotin Synthase Research Articles

A SNARE-Like Protein and Biotin Are Implicated in Soybean Cyst Nematode Virulence.

Phytoparasitic nematodes that are able to infect and reproduce on plants that are considered resistant are referred to as virulent. The mechanism(s) that virulent nematodes employ to evade or suppress host plant defenses are not well understood. Here we report the use of a genetic strategy (allelic imbalance analysis) to associate single nucleotide polymorphisms (SNPs) with nematode virulence genes in Heterodera glycines, the soybean cyst nematode (SCN). To accomplish this analysis, a custom SCN SNP array was developed and used to genotype SCN F3-derived populations grown on resistant and susceptible soybean plants. Three SNPs reproducibly showed allele imbalances between nematodes grown on resistant and susceptible plants. Two candidate SCN virulence genes that were tightly linked to the SNPs were identified. One SCN gene encoded biotin synthase (HgBioB), and the other encoded a bacterial-like protein containing a putative SNARE domain (HgSLP-1). The two genes mapped to two different linkage groups. HgBioB contained sequence polymorphisms between avirulent and virulent nematodes. However, the gene encoding HgSLP-1 had reduced copy number in virulent nematode populations and appears to produce multiple forms of the protein via intron retention and alternative splicing. We show that HgSLP-1 is an esophageal-gland protein that is secreted by the nematode during plant parasitism. Furthermore, in bacterial co-expression experiments, HgSLP-1 co-purified with the SCN resistance protein Rhg1 α-SNAP, suggesting that these two proteins physically interact. Collectively our data suggest that multiple SCN genes are involved in SCN virulence, and that HgSLP-1 may function as an avirulence protein and when absent it helps SCN evade host defenses.

Density functional theory calculations on the active site of biotin synthase: mechanism of S transfer from the Fe(2)S(2) cluster and the role of 1st and 2nd sphere residues.

Density functional theory (DFT) calculations are performed on the active site of biotin synthase (BS) to investigate the sulfur transfer from the Fe(2)S(2) cluster to dethiobiotin (DTB). The active site is modeled to include both the 1st and 2nd sphere residues. Molecular orbital theory considerations and calculation on smaller models indicate that only an S atom (not S²⁻) transfer from an oxidized Fe(2)S(2) cluster leads to the formation of biotin from the DTB using two adenosyl radicals generated from S-adenosyl-L-methionine. The calculations on larger protein active site model indicate that a 9-monothiobiotin bound reduced cluster should be an intermediate during the S atom insertion from the Fe(2)S(2) cluster consistent with experimental data. The Arg260 bound to Fe1, being a weaker donor than cysteine bound to Fe(2), determines the geometry and the electronic structure of this intermediate. The formation of this intermediate containing the C9-S bond is estimated to have a ΔG(≠) of 17.1 kcal/mol while its decay by the formation of the 2nd C6-S bond is calculated to have a ΔG(≠) of 29.8 kcal/mol, i.e. the 2nd C-S bond formation is calculated to be the rate determining step in the cycle and it leads to the decay of the Fe(2)S(2) cluster. Significant configuration interaction (CI), present in these transition states, helps lower the barrier of these reactions by ~30-25 kcal/mol relative to a hypothetical outer-sphere reaction. The conserved Phe285 residue near the Fe(2)S(2) active site determines the stereo selectivity at the C6 center of this radical coupling reaction. Reaction mechanism of BS investigated using DFT calculations. Strong CI and the Phe285 residue control the kinetic rate and stereochemistry of the product.

Paramagnetic Intermediates Generated by Radical S-Adenosylmethionine (SAM) Enzymes

ConspectusA [4Fe–4S]+ cluster reduces a bound S-adenosylmethionine (SAM) molecule, cleaving it into methionine and a 5′-deoxyadenosyl radical (5′-dA•). This step initiates the varied chemistry catalyzed by each of the so-called radical SAM enzymes. The strongly oxidizing 5′-dA• is quenched by abstracting a H-atom from a target species. In some cases, this species is an exogenous molecule of substrate, for example, l-tyrosine in the [FeFe] hydrogenase maturase, HydG. In other cases, the target is a proteinaceous residue as in all the glycyl radical forming enzymes. The generation of this initial radical species and the subsequent chemistry involving downstream radical intermediates is meticulously controlled by the enzyme so as to prevent unwanted reactions. But the manner in which this control is exerted is unknown. Electron paramagnetic resonance (EPR) spectroscopy has proven to be a valuable tool used to gain insight into these mechanisms.In this Account, we summarize efforts to trap such radical intermediates in radical SAM enzymes and highlight four examples in which EPR spectroscopic results have shed significant light on the corresponding mechanism. For lysine 2,3-aminomutase, nearly each possible intermediate, from an analogue of the initial 5′-dA• to the product radical l-β-lysine, has been explored. A paramagnetic intermediate observed in biotin synthase is shown to involve an auxiliary [FeS] cluster whose bridging sulfide is a co-substrate for the final step in the biosynthesis of vitamin B7. In HydG, the l-tyrosine substrate is converted in unprecedented fashion to a 4-oxidobenzyl radical on the way to generating CO and CN– ligands for the [FeFe] cluster of hydrogenase. And finally, EPR has confirmed a mechanistic proposal for the antibiotic resistance protein Cfr, which methylates the unactivated sp2-hybridized C8-carbon of an adenosine base of 23S ribosomal RNA.These four systems provide just a brief survey of the ever-growing set of radical SAM enzymes. The diverse chemistries catalyzed by these enzymes make them an intriguing target for continuing study, and EPR spectroscopy, in particular, seems ideally placed to contribute to our understanding.

The rate‐limiting steps in radical‐mediated carbon‐sulfur bond formation catalyzed by biotin synthase (580.6)

Biotin synthase is an S‐adenosylmethionine (AdoMet or SAM) radical enzyme that catalyzes the substitution of sulfur for hydrogen in dethiobiotin, generating two new C‐S bonds in the thiophane ring of biotin. In a reaction shared with other members of the Radical SAM superfamily, biotin synthase uses a [4Fe‐4S] cluster to catalyze the reduction of SAM to methionine and a 5'‐deoxyadenosyl radical. This highly reactive radical abstracts a hydrogen atom from the C9 position of dethiobiotin, presumably generating a dethiobiotinyl radical, that is then quenched by a sulfide from a nearby [2Fe‐2S] cluster. Following exchange methionine and 5'‐deoxyadenosine for a second equivalent of SAM, a second reaction sequence focused on the C6 position completes the formation of the thiophane ring. Kinetic analysis of pre‐steady‐state enzyme turnover indicates that formation of the monothiolated intermediate, 9‐mercaptodethiobiotin, is significantly slower than the subsequent ring closure. Using kinetic isotope effect (KIE) analysis, we demonstrate that hydrogen atom abstraction from the C9 methyl group is the overall rate‐limiting step, with a burst phase KIE of ~35 ‐ 40. During in vitro steady‐state turnover, regeneration of the active enzyme through FeS cluster assembly becomes partially rate‐limiting, resulting in a partial masking of this isotope effect to a yield a KIE of ~5. Finally, feeding studies in E. coli with labeled dethiobiotin exhibit a large KIE, suggesting that FeS cluster assembly is not rate‐limiting in the presence of the ISC iron‐sulfur cluster assembly systems. The implications of the very large non‐classical isotope effect will be discussed in terms of enzyme evolution and the potential biotechnological applications for AdoMet radical enzymes.Grant Funding Source: Supported by NSF (MCB‐1244632 to JTJ)

Development of Biotin-Prototrophic and -Hyperauxotrophic Corynebacterium glutamicum Strains

To develop the infrastructure for biotin production through naturally biotin-auxotrophic Corynebacterium glutamicum, we attempted to engineer the organism into a biotin prototroph and a biotin hyperauxotroph. To confer biotin prototrophy on the organism, the cotranscribed bioBF genes of Escherichia coli were introduced into the C. glutamicum genome, which originally lacked the bioF gene. The resulting strain still required biotin for growth, but it could be replaced by exogenous pimelic acid, a source of the biotin precursor pimelate thioester linked to either coenzyme A (CoA) or acyl carrier protein (ACP). To bridge the gap between the pimelate thioester and its dedicated precursor acyl-CoA (or -ACP), the bioI gene of Bacillus subtilis, which encoded a P450 protein that cleaves a carbon-carbon bond of an acyl-ACP to generate pimeloyl-ACP, was further expressed in the engineered strain by using a plasmid system. This resulted in a biotin prototroph that is capable of the de novo synthesis of biotin. On the other hand, the bioY gene responsible for biotin uptake was disrupted in wild-type C. glutamicum. Whereas the wild-type strain required approximately 1 μg of biotin per liter for normal growth, the bioY disruptant (ΔbioY) required approximately 1 mg of biotin per liter, almost 3 orders of magnitude higher than the wild-type level. The ΔbioY strain showed a similar high requirement for the precursor dethiobiotin, a substrate for bioB-encoded biotin synthase. To eliminate the dependency on dethiobiotin, the bioB gene was further disrupted in both the wild-type strain and the ΔbioY strain. By selectively using the resulting two strains (ΔbioB and ΔbioBY) as indicator strains, we developed a practical biotin bioassay system that can quantify biotin in the seven-digit range, from approximately 0.1 μg to 1 g per liter. This bioassay proved that the engineered biotin prototroph of C. glutamicum produced biotin directly from glucose, albeit at a marginally detectable level (approximately 0.3 μg per liter).

Developing a biological reducing system for use at elevated temperatures

S‐Adenosylmethionine (AdoMet)‐dependent radical enzymes constitute a rapidly expanding family of enzymes, the Radical SAM superfamily, that are involved in a variety of biological pathways. A defining characteristic of AdoMet‐dependent radical enzymes is the ability to use a reduced [4Fe‐4S] cluster to reductively cleave AdoMet, producing methionine and a 5′‐ deoxyadenosyl radical. This highly oxidizing radical intermediate initiates chemistry by abstracting a hydrogen atom from the substrate. Currently, the most common experimental means of reducing the [4Fe‐4S] cluster are the use of chemical reductants such as sodium dithionite, or the biochemical reductant flavodoxin. In the E. coli reducing system, NADPH reduces ferredoxin(flavodoxin) oxidoreductase (FNR), which then reduces flavodoxin (Fld) or ferredoxin (Fd). The E. coli system has drawbacks including thermal instability, and poor reaction kinetics. The development of a reducing system involving enzymes from thermophilic photosynthetic cyanobacteria will offer solutions to these problems, as such enzymes are stable and functional at elevated temperatures and exhibit rapid turnover due to their involvement in photosynthetic electron transport. We report that FNR from the thermophilic cyanobacterium, Thermosynechococcus elongatus, can be reduced by NADPH and rapidly transfers electrons to E. coli flavodoxin as well as T. elongatus ferredoxin. The component proteins are stable and active up to at least 65 °C. A heterologous reducing system comprised of NADPH, T. elongatus FNR, and E. coli flavodoxin is able to support the catalytic activity of biotin synthase from E. coli, as well as from thermophilic bacteria, including Methylococcus capsulatus. (Supported by NSF Grant MCB 09–23829 to J.T.J.)

Rickettsia felisinAedes albopictusMosquitoes, Libreville, Gabon

To the Editor: Rickettsia felis, an emerging pathogen first identified in the cat flea (1), has been detected in other fleas, ticks, mites, and booklice (2). R. felis can be cultured in mosquito cell lines derived from Anopheles gambiae and Aedes albopictus (Asian tiger) mosquitoes (2), so its compatibility with mosquitoes in nature can be suspected. In sub-Saharan Africa, R. felis bacteremia in humans is common¸ especially during the rainy season, when mosquitoes proliferate. We tested anthropophilic mosquitoes for the presence of R. felis DNA (3–5). During December 2008–January 2010, we randomly selected female Ae. albopictus and Ae. aegypti mosquitoes (96 each) from specimens obtained by human-landing collections from 4 sites in Libreville, Gabon (6). Specimens were collected during the rainy season (mid-January–end of May and end of September–mid-December); no parity data were available. We extracted 192 DNA samples from homogenate (abdomen, wings, legs) of each nonengorged, host-seeking, adult mosquito by using the BioRobot 8000 (QIAGEN S.A.S., Courtaboeuf, France) and QIAamp Media MDx Kit (QIAGEN) according to the manufacturer’s instructions. Samples were screened by quantitative real-time PCR (qPCR) targeting the biotin synthase (bioB) gene (4). Positive results were confirmed by qPCR-based molecular detection targeting the orfB gene, which codes for a transposition helper protein. This qPCR used a set of primers not previously used in our laboratory (R_fel.OrfB_F: 5′-CCCTTTTCGTAACGCTTTGCT-3′ and R_fel.OrfB_R: 5′-GGGCTAAACCAGGGAAACCT-3′) and the probe R_fel.OrfB_P: 6-FAM-TGTTCCGGTTTTAACGGCAGATACCCA-TAMRA. Specificity of the qPCR was tested in silico and on the 31 Rickettsia spp. from our laboratory. The final qPCR reaction mixture contained extracted DNA (5 μL) and mix (15 μL) that contained master mix (10 μL) from the QuantiTect Probe PCR Kit (QIAGEN, Hilden, Germany), each primer (0.5 μL, 20 pmol), probe (0.5 μL, 62.5 nmol), and RNase-free water (3.5 μL). Amplification and sequence detection were performed in a CFX96 Touch thermocycler (Bio-Rad, Marnes-la-Coquette, France) as follows:15 min at 95°C followed by 40 cycles of 1 s at 95°C, 40 s at 60°C, and 40 s at 45°C. Test results for all Ae. aegypti homogenates were negative for R. felis DNA. Of the 96 Ae. albopictus specimens, 3 (3.1%) had positive test results for the R. felis species–specific real-time qPCR and the confirmatory qPCR, with mean cycle thresholds ± SDs of 37.34 ± 1.7 (bioB gene; mean copies/mosquito 5 × 102 [minimum 1.2 × 102, maximum 1.4 × 103]) and 33.64 ± 1.4 (orfB gene; mean copies/mosquito 5 × 102 [minimum 1.5 × 102, maximum 1 × 103). One of the 3 samples was collected in January and 2 in March. The samples came from 3 different districts of Libreville (Akebe Poteau, Alibandeng, Camp des Boys) and were tested by nested PCR targeting the citrate synthase (gltA) gene (7). Rickettsia montanensis DNA was used as a positive control. Sequencing was performed as described (7), and ChromasPro version 1.34 (Technelysium Pty Ltd., Tewantin, Queensland, Australia) was used to analyze sequence data. Sequences of the bioB (120/120) and gltA (1,230/1,230) amplicons at the nucleotide level were 100% homologous to sequences for R. felis URRWXCal2 (GenBank accession no. {type:entrez-nucleotide,attrs:{text:C53,term_id:67003925,term_text:C53}}C53). The gltA fragment sequence was deposited in GenBank (accession no. {type:entrez-nucleotide,attrs:{text:JQ674484,term_id:386870494,term_text:JQ674484}}JQ674484). Mosquitoes were considered positive for R. felis when the qPCR result was 35 cycle thresholds for both genes. Contamination is a critical problem for the PCR-based identification of microbes. However, the validity of the data we report is based on strict laboratory procedures and controls that are commonly used in the World Health Organization Reference Center for Rickettsial Diseases, including rigorous positive and negative controls to validate the test. Each positive qPCR result was confirmed by another R. felis–specific qPCR (orfB) not previously used in our laboratory (to avoid contamination with other amplicons). Ae. albopictus mosquitos are native to Southeast Asia, colonizing rural and peri-urban sites. In Gabon, Ae. albopictus was the vector for outbreaks of chikungunya and dengue virus infections (6). Our study indicates that mosquitoes can carry R. felis, and the prevalence and load (1.8% –70% and 1.3 × 103–1.6 × 107, respectively) detected in mosquitoes in this study are consistent with the low-end range of those detected in cat fleas, the confirmed biological vector and reservoir (8,9). We investigated the presence of Rickettsia spp. in mosquitoes neglected as possible vectors of rickettsial diseases (2). Other Aedes spp. and other genera of mosquitoes should be tested. The role of mosquitoes as Rickettsia spp. vectors remains to be demonstrated in additional studies that use the Mitchell criteria. These studies should include the use of cell culture to isolate or detect R. felis in salivary glands of specimens from wild-caught mosquitoes, PCR, immunofluorescence, and the fluorescence in situ hybridization technique; demonstration of infection of a mosquito after experimental feeding on a bacteremic host or bacterial suspension; and demonstration of the transmission of bacteria to a vertebrate through the bite of a mosquito (10).

9-Mercaptodethiobiotin is generated as a ligand to the [2Fe-2S]+ cluster during the reaction catalyzed by biotin synthase from Escherichia coli.

Biotin synthase catalyzes formation of the thiophane ring through stepwise substitution of a sulfur atom for hydrogen atoms at the C9 and C6 positions of dethiobiotin. Biotin synthase is a radical S-adenosylmethionine (SAM) enzyme that reductively cleaves S-adenosylmethionine, generating 5'-deoxyadenosyl radicals that initially abstract a hydrogen atom from the C9 position of dethiobiotin. We have proposed that the resulting dethiobiotinyl radical is quenched by the μ-sulfide of the nearby [2Fe-2S](2+) cluster, resulting in coupled formation of 9-mercaptodethiobiotin and a reduced [2Fe-2S](+) cluster. This reduced FeS cluster is observed by electron paramagnetic resonance spectroscopy as a mixture of two orthorhombic spin systems. In the present work, we use isotopically labeled 9-mercaptodethiobiotin and enzyme to probe the ligand environment of the [2Fe-2S](+) cluster in this reaction intermediate. Hyperfine sublevel correlation spectroscopy (HYSCORE) spectra exhibit strong cross-peaks demonstrating strong isotropic coupling of the nuclear spin with the paramagnetic center. The hyperfine coupling constants are consistent with a structural model for the reaction intermediate in which 9-mercaptodethiobiotin is covalently coordinated to the remnant [2Fe-2S](+) cluster.

Genome-Wide Analysis of Biotin Biosynthesis in Eukaryotic Photosynthetic Algae

Biotin is a cofactor responsible for carbon dioxide transfer in several carboxylase enzymes, which play a significant role in various metabolic reactions such as fatty acid synthesis, branched chain amino acid catabolism, and gluconeogenesis. Biotin is also involved in citric acid cycle, which is the process of biochemical energy generation during aerobic respiration. Though the function of biotin in the growth of algae has been extensively investigated, little is known about the biosynthetic routes of biotin in the algal kingdom. In the present study, 44 biotin biosynthesis-related genes were identified from 14 eukaryotic photosynthetic algal genomes by BLASTP and TBLASN programs. A comprehensive analysis was performed to characterize distribution, phylogeny, structure domains, and coevolution patterns of those genes. Forty-four biotin biosynthesis-related enzymes (BBREs) were found to be distributed in three groups: 7-keto-8-aminopelargonic acid synthase, diaminopelargonic acid synthase/dethiobiotin synthetase, and biotin synthase. Structure domains were considerably conserved among the subfamilies of BBREs. The intramolecular coevolutionary sites are widely distributed in biotin synthase. The present study provides new insights into the origin and evolution of biotin biosynthetic pathways in eukaryotic photosynthetic algae. Furthermore, the characterization of biotin biosynthesis-related genes from algae will promote the identification and functional studies of BBREs.

Reduction of the [2Fe–2S] Cluster Accompanies Formation of the Intermediate 9-Mercaptodethiobiotin in Escherichia coli Biotin Synthase

Biotin synthase catalyzes the conversion of dethiobiotin (DTB) to biotin through the oxidative addition of sulfur between two saturated carbon atoms, generating a thiophane ring fused to the existing ureido ring. Biotin synthase is a member of the radical SAM superfamily, composed of enzymes that reductively cleave S-adenosyl-l-methionine (SAM or AdoMet) to generate a 5'-deoxyadenosyl radical that can abstract unactivated hydrogen atoms from a variety of organic substrates. In biotin synthase, abstraction of a hydrogen atom from the C9 methyl group of DTB would result in formation of a dethiobiotinyl methylene carbon radical, which is then quenched by a sulfur atom to form a new carbon-sulfur bond in the intermediate 9-mercaptodethiobiotin (MDTB). We have proposed that this sulfur atom is the μ-sulfide of a [2Fe-2S](2+) cluster found near DTB in the enzyme active site. In the present work, we show that formation of MDTB is accompanied by stoichiometric generation of a paramagnetic FeS cluster. The electron paramagnetic resonance (EPR) spectrum is modeled as a 2:1 mixture of components attributable to different forms of a [2Fe-2S](+) cluster, possibly distinguished by slightly different coordination environments. Mutation of Arg260, one of the ligands to the [2Fe-2S] cluster, causes a distinctive change in the EPR spectrum. Furthermore, magnetic coupling of the unpaired electron with (14)N from Arg260, detectable by electron spin envelope modulation (ESEEM) spectroscopy, is observed in WT enzyme but not in the Arg260Met mutant enzyme. Both results indicate that the paramagnetic FeS cluster formed during catalytic turnover is a [2Fe-2S](+) cluster, consistent with a mechanism in which the [2Fe-2S](2+) cluster simultaneously provides and oxidizes sulfide during carbon-sulfur bond formation.

ChemInform Abstract: Catalytic Functions of Cubane‐Type M4S4 Clusters

AbstractReview: 91 refs.

Generation of adenosyl radical from S-adenosylmethionine (SAM) in biotin synthase

A mechanism of the C―S bond activation of S-adenosylmethionine (SAM) in biotin synthase is discussed from quantum mechanical/molecular mechanical (QM/MM) computations. The active site of the enzyme involves a [4Fe–4S] cluster, which is coordinated to the COO − and NH 2 groups of the methionine moiety of SAM. The unpaired electrons on the iron atoms of the [4Fe–4S] 2+ cluster are antiferromagnetically coupled, resulting in the S = 0 ground spin state. An electron is transferred from an electron donor to the [4Fe–4S] 2+–SAM complex to produce the catalytically active [4Fe–4S] + state. The SOMO of the [4Fe–4S] +–SAM complex is localized on the [4Fe–4S] moiety and the spin density of the [4Fe–4S] core is calculated to be 0.83. The C―S bond cleavage is associated with the electron transfer from the [4Fe–4S] + cluster to the antibonding σ* C―S orbital. The electron donor and acceptor states are effectively coupled with each other at the transition state for the C―S bond cleavage. The activation barrier is calculated to be 16.0 kcal/mol at the QM (B3LYP/SV(P))/MM (CHARMm) level of theory and the C―S bond activation process is 17.4 kcal/mol exothermic, which is in good agreement with the experimental observation that the C―S bond is irreversibly cleaved in biotin synthase. The sulfur atom of the produced methionine molecule is unlikely to bind to an iron atom of the [4Fe–4S] 2+ cluster after the C―S bond cleavage from the energetical and structural points of view.

Structural Basis for Reductive Radical Formation and Electron Recycling in (R)-2-Hydroxyisocaproyl-CoA Dehydratase

The radical enzyme (R)-2-hydroxyisocaproyl-CoA dehydratase catalyzes the dehydration of (R)-2-hydroxyisocaproyl-CoA in the fermentation of l-leucine by the human pathogenic bacterium Clostridium difficile. In contrast to other radical enzymes, such as bacterial class II ribonucleotide reductase or biotin synthase, the Fe/S cluster containing (R)-2-hydroxyisocaproyl-CoA dehydratase requires no special cofactors such as coenzyme B(12) or S-adenosylmethionine for radical generation. Instead it uses a single high-energy electron that is recycled after each turnover. The catalyzed reaction, an atypical α/β-dehydration, depends on the reductive formation of ketyl radicals on the substrate generated by injection of a single electron from the ATP-dependent activator protein. So far, it is unknown how the active electron is recycled and how unwanted side reactions are prevented, allowing for up to 10,000 turnovers. The crystal structure reveals that the heterodimeric protein contains two [4Fe-4S] clusters at a distance of 12 Å, each coordinated by three cysteines and one terminal ligand. The cluster in the α-subunit is part of the active site. In the absence of substrate, a water/hydroxide ion acts as the fourth ligand. The substrate replaces this ligand and coordinates the cluster via the carbonyl-oxygen of the thioester group. The cluster in the β-subunit has a terminal sulfhydryl/sulfido ligand and can act as a reservoir to protect the electron from unwanted side reactions via a recycling mechanism. The crystal structure of (R)-2-hydroxyisocaproyl-CoA dehydratase serves as a model for the reductively radical-generating metalloenzymes of the (R)-2-hydroxyacyl-CoA dehydratase and benzoyl-CoA reductase families.

Catalytic functions of cubane-type M4S4 clusters

Catalysis by the cubane-type Fe4S4 cluster has been found in metalloproteins such as biotin synthase, aconitase, and IspH, whereas some synthetic M4S4 clusters show catalytic activities for biologically related reactions or various types of organic transformations. This minireview summarizes recent progress in the chemistry of catalytic functions of cubane-type M4S4 clusters.

Uncovering the Human Methyltransferasome

We present a comprehensive analysis of the human methyltransferasome. Primary sequences, predicted secondary structures, and solved crystal structures of known methyltransferases were analyzed by hidden Markov models, Fisher-based statistical matrices, and fold recognition prediction-based threading algorithms to create a model, or profile, of each methyltransferase superfamily. These profiles were used to scan the human proteome database and detect novel methyltransferases. 208 proteins in the human genome are now identified as known or putative methyltransferases, including 38 proteins that were not annotated previously. To date, 30% of these proteins have been linked to disease states. Possible substrates of methylation for all of the SET domain and SPOUT methyltransferases as well as 100 of the 131 seven-β-strand methyltransferases were surmised from sequence similarity clusters based on alignments of the substrate-specific domains.

Biotin Synthase Exhibits Burst Kinetics and Multiple Turnovers in the Absence of Inhibition by Products and Product-Related Biomolecules

Biotin synthase (BS) is a member of the "SAM radical" superfamily of enzymes, which catalyze reactions in which the reversible or irreversible oxidation of various substrates is coupled to the reduction of the S-adenosyl-l-methionine (AdoMet) sulfonium to generate methionine and 5'-deoxyadenosine (dAH). Prior studies have demonstrated that these products are modest inhibitors of BS and other members of this enzyme family. In addition, the in vivo catalytic activity of Escherichia coli BS requires expression of 5'-methylthioadenosine/S-adenosyl-l-homocysteine nucleosidase, which hydrolyzes 5'-methylthioadenosine (MTA), S-adenosyl-l-homocysteine (AdoHcy), and dAH. In the present work, we confirm that dAH is a modest inhibitor of BS (K(i) = 20 μM) and show that cooperative binding of dAH with excess methionine results in a 3-fold enhancement of this inhibition. However, with regard to the other substrates of MTA/AdoHcy nucleosidase, we demonstrate that AdoHcy is a potent inhibitor of BS (K(i) ≤ 650 nM) while MTA is not an inhibitor. Inhibition by both dAH and AdoHcy likely accounts for the in vivo requirement for MTA/AdoHcy nucleosidase and may help to explain some of the experimental disparities between various laboratories studying BS. In addition, we examine possible inhibition by other AdoMet-related biomolecules present as common contaminants in commercial AdoMet preparations and/or generated during an assay, as well as by sinefungin, a natural product that is a known inhibitor of several AdoMet-dependent enzymes. Finally, we examine the catalytic activity of BS with highly purified AdoMet in the presence of MTAN to relieve product inhibition and present evidence suggesting that the enzyme is half-site active and capable of undergoing multiple turnovers in vitro.

Overexpression of biotin synthase and biotin ligase is required for efficient generation of sulfur-35 labeled biotin in E. coli

BackgroundBiotin is an essential enzyme cofactor that acts as a CO2 carrier in carboxylation and decarboxylation reactions. The E. coli genome encodes a biosynthetic pathway that produces biotin from pimeloyl-CoA in four enzymatic steps. The final step, insertion of sulfur into desthiobiotin to form biotin, is catalyzed by the biotin synthase, BioB. A dedicated biotin ligase (BirA) catalyzes the covalent attachment of biotin to biotin-dependent enzymes. Isotopic labeling has been a valuable tool for probing the details of the biosynthetic process and assaying the activity of biotin-dependent enzymes, however there is currently no established method for 35S labeling of biotin.ResultsIn this study, we produced [35S]-biotin from Na35SO4 and desthiobiotin with a specific activity of 30.7 Ci/mmol, two orders of magnitude higher than previously published methods. The biotinylation domain (PfBCCP-79) from the Plasmodium falciparum acetyl-CoA carboxylase (ACC) was expressed in E. coli as a biotinylation substrate. We found that overexpression of the E. coli biotin synthase, BioB, and biotin ligase, BirA, increased PfBCCP-79 biotinylation 160-fold over basal levels. Biotinylated PfBCCP-79 was purified by affinity chromatography, and free biotin was liberated using acid hydrolysis. We verified that we had produced radiolabeled biologically active [D]-biotin that specifically labels biotinylated proteins through reuptake in E. coli.ConclusionsThe strategy described in our report provides a simple and effective method for the production of [35S]-biotin in E. coli based on affinity chromatography.

Evaluation of biosynthetic pathways for the unique dithiolate ligand of the FeFe hydrogenase H-cluster

FeFe hydrogenases [1] catalyze the reversible oxidation of dihydrogen at a remarkable rate [2, 3]. Their active site, termed the H-cluster, comprises a classic [4Fe–4S] cubane that is covalently linked via a bridging cysteine residue to a biologically unprecedented organometallic binuclear 2Fesubcluster. The two clusters are electronically [4] and magnetically [5] inseparable. The low-spin and lowvalence iron centers of the 2Fe-subcluster are stabilized by diatomic carbonyl (CO) and cyanide (CN) strong-field ligands and they are bridged by a dithiolate group (SCH2XCH2S) , whose central atom (bridgehead) has not yet been unequivocally determined. On the basis of X-ray crystallography, Fourier transform IR spectroscopy, N hyperfine sublevel correlation spectroscopy, and theoretical calculations, the dithiolate ligand has been proposed to be 1,3-propanedithiolate (PDT; X is CH2) [6], dithiomethylamine (DTMA; X is NH) [7, 8], or dithiomethyl ether (DTME; X is O) [9]. This unprecedented coordination environment of the 2Fe-subcluster has stimulated an intensive biochemical research to identify the source and the exact biosynthetic pathway leading to the formation of these biologically unique ligands. However, the proposed hypotheses regarding the biosynthesis of the dithiolate bridging ligand are based only on chemical intuition [10, 11]. Therefore, in this communication we report a density-functional-theorybased, comparative evaluation of the energetic and mechanistic feasibility of various pathways. For the sake of completeness, all three possible candidates of dithiolates (X is CH2, NH, or O) were examined. Staying within the boundaries of known biochemical processes, we have found that a pathway that includes known S-adenosylmethionine (SAM)-catalyzed reactions is both energetically and mechanistically more favorable. Although we describe the pathway for the formation of PDT, it can be readily adopted for the biosynthesis of the DTMA and DTME analogues, provided that the proper precursor molecule is available. The enzymes HydE, HydF, and HydG have been identified as necessary and likely sufficient for the biosynthesis of the 2Fe-subcluster of the H-cluster. HydF shows GTPase activity and serves as the scaffold protein where the 2Fesubcluster is assembled [12, 13]. HydE and HydG belong to the radical-SAM superfamily of enzymes [14]. These enzymes generate the highly reactive 5-adenosyl radical (50-dA!) that can drive a variety of biochemical processes, including hydrogen atom abstraction, carbon–carbon bond cleavage, and sulfur insertion [15]. A biosynthetic hypothesis suggested that glycine could serve as the source of the diatomic ligands and it was supported by a computational investigation that is conceptually similar to the one presented here [10]. This hypothesis was recently experimentally verified, as the presence of CO and CN was detected when HydG was treated with tyrosine, through a mechanism that likely involves the formation of a glycyl radical [16, 17]. Therefore, HydE is most likely responsible for the biosynthesis of the dithiolate bridge on a [2Fe–2S] cluster located in the scaffold protein HydF. This [2Fe–2S] cluster is coordinated by three highly conserved cysteine residues, whereas the fourth ligand is readily exchangeable [13]. Moreover, the radical-SAM enzymes biotin synthase Electronic supplementary material The online version of this article (doi:10.1007/s00775-010-0698-y) contains supplementary material, which is available to authorized users.

ChemInform Abstract: The Insertion of Sulfur into the Non-Activated C-H Bond. A Possible Model for Penicillin Cyclase and Biotin Synthase.

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A Complex between Biotin Synthase and the Iron−Sulfur Cluster Assembly Chaperone HscA That Enhances in Vivo Cluster Assembly

Biotin synthase (BioB) is an iron-sulfur enzyme that catalyzes the last step in biotin biosynthesis, the insertion of sulfur between the C6 and C9 atoms of dethiobiotin to complete the thiophane ring of biotin. Recent in vitro experiments suggest that the sulfur is derived from a [2Fe-2S](2+) cluster within BioB, and that the remnants of this cluster dissociate from the enzyme following each turnover. For BioB to catalyze multiple rounds of biotin synthesis, the [2Fe-2S](2+) cluster in BioB must be reassembled, a process that could be conducted in vivo by the ISC or SUF iron-sulfur cluster assembly systems. The bacterial ISC system includes HscA, an Hsp70 class molecular chaperone, whose yeast homologue has been shown to play an important but nonessential role in assembly of mitochondrial FeS clusters in Saccharomyces cerevisiae. In this work, we show that in Escherichia coli, HscA significantly improves the efficiency of the in vivo assembly of the [2Fe-2S](2+) cluster on BioB under conditions of low to moderate iron. In vitro, we show that HscA binds with increased affinity to BioB missing one or both FeS clusters, with a maximum of two HscA molecules per BioB dimer. BioB binds to HscA in an ATP/ADP-independent manner, and a high-affinity complex is also formed with a truncated form of HscA that lacks the nucleotide binding domain. Further, the BioB-HscA complex binds the FeS cluster scaffold protein IscU in a noncompetitive manner, generating a complex that contains all three proteins. We propose that HscA plays a role in facilitating the transfer of FeS clusters from IscU into the appropriate target apoproteins such as biotin synthase, perhaps by enhancing or prolonging the requisite protein-protein interaction.