Olaf Burghaus

The radical SAM enzyme AlbA catalyzes thioether bond formation in subtilosin A

Subtilosin A is a 35-residue, ribosomally synthesized bacteriocin encoded by the sbo-alb operon of Bacillus subtilis. It is composed of a head-to-tail circular peptide backbone that is additionally restrained by three unusual thioether bonds between three cysteines and the α-carbon of one threonine and two phenylalanines, respectively. In this study, we demonstrate that these bonds are synthesized by the radical S-adenosylmethionine enzyme AlbA, which is encoded by the sbo-alb operon and comprises two [4Fe-4S] clusters. One [4Fe-4S] cluster is coordinated by the prototypical CXXXCXXC motif and is responsible for the observed S-adenosylmethionine cleavage reaction, whereas the second [4Fe-4S] cluster is required for the generation of all three thioether linkages. On the basis of the obtained results, we propose a new radical mechanism for thioether bond formation. In addition, we show that AlbA-directed substrate transformation is leader-peptide dependent, suggesting that thioether bond formation is the first step during subtilosin A maturation.

SufU Is an Essential Iron-Sulfur Cluster Scaffold Protein in Bacillus subtilis

Bacteria use three distinct systems for iron-sulfur (Fe/S) cluster biogenesis: the ISC, SUF, and NIF machineries. The ISC and SUF systems are widely distributed, and many bacteria possess both of them. In Escherichia coli, ISC is the major and constitutive system, whereas SUF is induced under iron starvation and/or oxidative stress. Genomic analysis of the Fe/S cluster biosynthesis genes in Bacillus subtilis suggests that this bacteriums genome encodes only a SUF system consisting of a sufCDSUB gene cluster and a distant sufA gene. Mutant analysis of the putative Fe/S scaffold genes sufU and sufA revealed that sufU is essential for growth under minimal standard conditions, but not sufA. The drastic growth retardation of a conditional mutant depleted of SufU was coupled with a severe reduction of aconitase and succinate dehydrogenase activities in total-cell lysates, suggesting a crucial function of SufU in Fe/S protein biogenesis. Recombinant SufU was devoid of Fe/S clusters after aerobic purification. Upon in vitro reconstitution, SufU bound an Fe/S cluster with up to approximately 1.5 Fe and S per monomer. The assembled Fe/S cluster could be transferred from SufU to the apo form of isopropylmalate isomerase Leu1, rapidly forming catalytically active [4Fe-4S]-containing holo-enzyme. In contrast to native SufU, its D43A variant carried a Fe/S cluster after aerobic purification, indicating that the cluster is stabilized by this mutation. Further, we show that apo-SufU is an activator of the cysteine desulfurase SufS by enhancing its activity about 40-fold in vitro. SufS-dependent formation of holo-SufU suggests that SufU functions as an Fe/S cluster scaffold protein tightly cooperating with the SufS cysteine desulfurase.

Two [4Fe-4S] Clusters Containing Radical SAM Enzyme SkfB Catalyze Thioether Bond Formation during the Maturation of the Sporulation Killing Factor

The sporulation killing factor (SKF) is a 26-residue ribosomally assembled and posttranslationally modified sactipeptide. It is produced by Bacillus subtilis 168 and plays a key role in its sporulation. Like all sactipeptides, SKF contains a thioether bond, which links the cysteine residue Cys4 with the α-carbon of the methionine residue Met12. In this study we demonstrate that this bond is generated by the two [4Fe-4S] clusters containing radical SAM enzyme SkfB, which is encoded in the skf operon. By mutational analysis of both cluster-binding sites, we were able to postulate a mechanism for thioether generation which is in agreement with that of AlbA. Furthermore, we were able to show that thioether bond formation is specific toward hydrophobic amino acids at the acceptor site. Additionally we demonstrate that generation of the thioether linkage is leader-peptide-dependent, suggesting that this reaction is the first step in SKF maturation.

The PqqD homologous domain of the radical SAM enzyme ThnB is required for thioether bond formation during thurincin H maturation

Thurincin H is a 31‐residue, ribosomally synthesized bacteriocin originating from the thn operon of Bacillus thuringiensis SF361. It is the only known sactipeptide carrying four thioether bridges between four cysteines and the α‐carbons of a serine, an asparagine and two threonine residues. By analysis of the thn operon and use of in vitro studies we now reveal that ThnB is a radical S‐adenosylmethionine (SAM) enzyme containing two [4Fe–4S] clusters. Furthermore, we confirm the involvement of ThnB in the formation of the thioether bonds present within the structure of thurincin H. Finally, we show that the PqqD homologous N‐terminal domain of ThnB is essential for maturation of the thurincin H precursor peptide, but not for the SAM cleavage activity of ThnB.

Iron 10‐Thiacorroles: Bioinspired Iron(III) Complexes with an Intermediate Spin (S=3/2) Ground State

A first systematic study upon the preparation and exploration of a series of iron 10-thiacorroles with simple halogenido (F, Cl, Br, I), pseudo-halogenido (N3 , I3 ) and solvent-derived axial ligands (DMSO, pyridine) is reported. The compounds were prepared from the free-base octaethyl-10-thiacorrole by iron insertion and subsequent ligand-exchange reactions. The small N4 cavity of the ring-contracted porphyrinoid results in an intermediate spin (i.s., S=3/2) state as the ground state for the iron(III) ion. In most of the investigated cases, the i.s. state is found unperturbed and independent of temperature, as determined by a combination of X-ray crystallography and magnetometry with (1) H NMR-, EPR-, and Mössbauer spectroscopy. Two exceptions were found. The fluorido iron(III) complex is inhomogenous in the solid and contains a thermal i.s. (S=3/2)→high spin (h.s., S=5/2) crossover fraction. On the other side, the cationic bis(pyridine) complex resides in the expected low spin (l.s., S=1/2) state. Chemically, the iron 10-thiacorroles differ from the iron porphyrins mainly by weaker axial ligand binding and by a cathodic shift of the redox potentials. These features make the 10-thiacorroles interesting ligands for future research on biomimetic catalysts and model systems for unusual heme protein active sites.

The Frataxin Homologue Fra Plays a Key Role in Intracellular Iron Channeling in Bacillus subtilis

Frataxin homologues are important iron chaperones in eukarya and prokarya. Using a native proteomics approach we were able to identify the structural frataxin homologue Fra (formerly YdhG) of Bacillus subtilis and to quantify its native iron‐binding stoichiometry. Using recombinant proteins we could show in vitro that Fra is able to transfer iron onto the B. subtilis SUF system for iron–sulfur cluster biosynthesis. In a four‐constituents reconstitution system (including SufU, SufS, Fra and CitB) we observed a Fra‐dependent formation of a [4 Fe–4 S] cluster on SufU that could be efficiently transferred onto the target apo‐aconitase (CitB). A Δfra deletion mutant showed a severe growth phenotype associated with a broadly disturbed iron homeostasis; this indicates that Fra is a central component of intracellular iron channeling in B. subtilis.

Double-Bond Isomerization: Highly Reactive Nickel Catalyst Applied in the Synthesis of the Pheromone (9Z,12Z)-Tetradeca-9,12-dienyl Acetate

A highly reactive nickel catalyst comprising NiCl2(dppp) or NiCl2(dppe) with zinc powder, ZnI2 and Ph2PH, was applied in the isomerization of terminal alkenes to Z-2-alkenes. The double-bond geometry of the 2-alkene can be controlled via the reaction temperature to yield the 2-Z-alkenes in excellent yields and high Z-selectivities. The formation of other constitutional isomers, such as 3-alkenes, is suppressed on the basis of the proposed mechanism via a 1,2-hydride shift from the metal to the Ph2P ligand. The nickel-catalyzed isomerization reaction was then applied in the synthesis of (9Z,12Z)-tetradeca-9,12-dienyl acetate, a pheromone with a 2Z,5Z-diene subunit.

Nonlinear ion transport in the supercooled ionic liquid 1-hexyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide: Frequency dependence of third-order and fifth-order conductivity coefficients.

We have carried out nonlinear ion conductivity measurements on the supercooled ionic liquid 1-hexyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide[C6mim][NTf2] by applying ac electric fields with amplitudes up to about 200 kV/cm. At these field amplitudes, 3ω and 5ω harmonic components in the current response were detected, and the higher-order conductivity coefficients σ3 (1),σ3 (3),σ5 (3), and σ5 (5) were determined. The frequency and temperature dependence of these conductivity coefficients was analyzed in detail. The most important findings were the following: (i) The third-order spectra σ3 (1) and σ3 (3) exhibit very similar values in the dc plateau regime but differ considerably in the dispersive regime. The same was observed for the fifth order spectra σ5 (3) and σ5 (5). (ii) In the dispersive regime, the third-order spectra display a minimum, while the fifth-order spectra display a maximum. (iii) The frequencies of these minima and maxima are thermally activated with the same activation energy as the low-field dc conductivity σ1,dc, whereas the dc values of the higher-order conductivity coefficients, σ3,dc and σ5,dc, are characterized by lower activation energies.

1,2,4,5-Tetra([5]trovacenyl)benzene: an organometallic tetraradical displaying pronounced electro- and magnetocommunication

The organometallic tetraradical 1,2,4,5-[(eta)7-C7H7)V(all eta]5-C5H4)]4C6H2 has been prepared and structurally characterized. The isotropic EPR spectrum displays 29 a(51V) hyperfine lines, the intensity distribution slightly deviating from binomial. Exchange coupling therefore approaches the strong exchange limit, J(ortho) not equal to J(meta) not equal to J(para) greater or approximately 50 a(51V) with a(51V)= 0.0067 cm(-1). According to magnetic susceptometry, the interaction is antiferromagnetic. While redox splittings deltaE(1/2) are resolved for the four reduction steps this is not the case for oxidation.

Zinn in der Peripherie von Bis(aren)metall-Komplexen des Vanadiums und Chroms

Durch Metallatom-Ligand-Cokondensation sowie uber naschemische Verfahren (Lithiierung und Folgereaktion) wurden als erste organostannylsubstituierte Bis(aren)metall-Komplexe die Verbindungen (R3Sn-η6-C6H5)2M dargestellt: 15 (R = Me, M = V), 16 (R = Ph, M = V), 13 (R = Me, M = Cr), 17 (R = Ph, M = Cr). Trotz der sperrigen Natur des Ph3Sn-Substituenten weicht die Geometrie der zentralen Sandwicheinheit in 17 nur unwesentlich von der des unsubstituierten Grundkorpers (C6H6)2Cr (2) ab. Die trikline Elementarzelle von 17 (Raumgruppe P1; a = 9.414(4), b = 9.877(5), c = 11.012(13) A; α = 83.51(7), β = 87.95(7), γ = 72.67(4)°) enthalt ein unabhangiges Molekul. Organostannylgruppen storen die Elektronenstruktur der Bis(aren)metall-Einheit offenbar nur geringfugig, denn die EPR-Spektren der M(d5) Spezies lassen keine Abweichungen von axialer Symmetrie erkennen. Die Potentiale fur die reversible Oxidation der Me3Sn-substituierten Komplexe 13 und 15 weichen nur wenig (anodische Verschiebung ≤ 20 mV) von denen der Grundkorper 1 und 2 ab, die Reduktion ist in beiden Fallen irreversibel. Grosere anodische Verschiebungen weisen die Ph3Sn-Derivate 16 und 17 auf; auch fur diese sind nur die Redoxpaare 0/+ reversibel. Der Resistenz der Neutralkomplexe in protischen Medien steht die uberaus leicht erfolgende Hydrodestannylierung der Komplexkationen gegenuber. Durch Ummetallierung wird aus 13 (Li-η6-C6H5)2Cr hilfsbasenfrei und streng 1,1′-dimetalliert gewonnen. Metal π Complexes of Benzene Derivatives. 53 [1] Tin in the Periphery of Bis(arene)metal Complexes of Vanadium and Chromium By means of metal-atom ligand-vapor cocondensation as well as via wet chemical methods (lithiation and follow-up reaction) the first organostannyl substituted bis(arene)metal complexes (R3Sn-η6-C6H5)2M have been prepared: 15 (R = Me, M = V), 16 (R = Ph, M = V), 13 (R = Me, M = V), 17 (R = Ph, M = Cr). Despite the bulkiness of the Ph3Sn groups the geometry of the central sandwich unit in 17 deviates only marginally from that of the parent complex (C6H6)2Cr (2). The triclinic unit cell of 17 (space group: P1; a = 9.414(4), b = 9.877(5), c = 11.012(13) A; α = 83.51(7), β = 87.95(7), γ = 72.67(4)°) contains one independent molecule. Perturbation of the electronic structure of the bis(arene)metal unit by organostannyl groups appears to be minute because EPR spectra of the M(d5) species fail to reveal deviations from axial symmetry. The potentials for reversible oxidation of the Me3Sn-substituted complexes 13 and 15 differ insignificantly (anodic shifts ≤ 20 mV) from those of the parent species 1 and 2; reductions are irreversible in both cases. More sizeable anodic shifts are observed for the Ph3Sn-derivatives 16 and 17; here as well, only the redox pairs 0/+ are reversible. The resistance of the neutral complexes to protic media contrasts to ready hydrodestannylation of the complex cations. By way of metal exchange, employing n-butyl lithium, 13 affords (Li-η6-C6H5)2Cr strictly 1,1′-disubstituted and devoid of auxiliary base.