Partial structure and properties of the ferredoxin from Rhodymenia palmata

A ferredoxin of MW 11 000 was isolated from the marine alga Rhodymenia palmata (Palmaria palmata). In its oxidised form the ferredoxin had absorption maxima at 276, sh 281, 328, 423 and 465 nm, and contained a single [2Fe-2S] cluster. The midpoint potential of the ferredoxin was ?400 mV and it effectively mediated electron transport in NADP⁺-photoreduction by higher plant chloroplasts, and pyruvate decarboxylation by the phosphoroclastic system of an anacrobic bacterium. The amino acid composition was Lys₃, His₁, Arg₁, Asx₁₂, Thr₉, Ser₈, Glx₁₃, Pro₄, Gly₈, Ala₇, Cys₅, Val₈, Ile₄, Leu₉, Tyr₄, Phe₂; tryptophan and methionine were absent from the molecule. The N-terminal amino acid region consisting of ca half the total amino acid sequence was determined using an automatic sequencer.     

Extrahelical cytosine bases in DNA duplexes containing d[GCC]n·d[GCC]n repeats: detection by a mechlorethamine crosslinking reaction

The cytosine–cytosine (C–C) pair is one of the least stable DNA mismatch pairs. The bases of the C–C mismatch are only weakly hydrogen bonded, and previous work has shown that, in certain sequence contexts, they can become unstacked from the core helix, and adopt an ‘extrahelical’ location. Here, using DNA duplexes with d[GCC]_n·d[GCC]_n fragments containing C–C mismatches in a 1,4 bp relationship, we show that cytosine bases of different formal mismatch pairs can be crosslinked by mechlorethamine. For example, in the duplex d[CTCTCGCCGCCGCCGTATC]·d[GATACGCCGCCGCCGAGAG], where underlined cytosine bases are present as the formal C–C mismatch pairs C₇–C₃₂, C₁₀–C₂₉ and C₁₃–C₂₆, we show that two mechlorethamine crosslinks form between C₁₃ and C₂₉ and between C₁₀ and C₃₂, in addition to crosslinks at C₇–C₃₂, C₁₀–C₂₉ and C₁₃–C₂₆ (we have reported previously the crosslinking of formal C–C pairs by mechlorethamine). We interpret the formation of the C₁₃–C₂₉ and C₁₀–C₃₂ crosslinks as evidence of an extrahelical location of the crosslinkable cytosines. Such extrahelical cytosine bases have been observed previously for a single C–C mismatch pair (in the so-called E-motif conformation). In the E-motif, the extrahelical cytosines are folded back towards the 5′-end of the duplex, consistent with our crosslinking data, and also consistent with the absence of C₇–C₂₉ and C₁₀–C₂₆ crosslinks in the current work. Hence, our data provide evidence for an extended E-motif DNA (eE-DNA) conformation in short d[GCC]_n·d[GCC]_n repeat fragments, and raise the possibility that such structures might occur in much longer d[GCC]_n·d[GCC]_n repeat tracts.     

Insights into the structure,function and evolution of the radical-SAM 23S rRNA methyltransferase Cfr that confers antibiotic resistance in bacteria

The Cfr methyltransferase confers combined resistance to five classes of antibiotics that bind to the peptidyl tranferase center of bacterial ribosomes by catalyzing methylation of the C-8 position of 23S rRNA nucleotide A2503. The same nucleotide is targeted by the housekeeping methyltransferase RlmN that methylates the C-2 position. Database searches with the Cfr sequence have revealed a large group of closely related sequences from all domains of life that contain the conserved CX₃CX₂C motif characteristic of radical S-adenosyl-l-methionine (SAM) enzymes. Phylogenetic analysis of the Cfr/RlmN family suggests that the RlmN subfamily is likely the ancestral form, whereas the Cfr subfamily arose via duplication and horizontal gene transfer. A structural model of Cfr has been calculated and used as a guide for alanine mutagenesis studies that corroborate the model-based predictions of a 4Fe–4S cluster, a SAM molecule coordinated to the iron–sulfur cluster (SAM1) and a SAM molecule that is the putative methyl group donor (SAM2). All mutations at predicted functional sites affect Cfr activity significantly as assayed by antibiotic susceptibility testing and primer extension analysis. The investigation has identified essential amino acids and Cfr variants with altered reaction mechanisms and represents a first step towards understanding the structural basis of Cfr activity.     

Determination of base and backbone contributions to the thermodynamics of premelting and melting transitions in B DNA

In previous papers of this series the temperature-dependent Raman spectra of poly(dA)·poly(dT) and poly(dA–dT)·poly(dA–dT) were used to characterize structurally the melting and premelting transitions in DNAs containing consecutive A·T and alternating A·T/T·A base pairs. Here, we describe procedures for obtaining thermodynamic parameters from the Raman data. The method exploits base-specific and backbone-specific Raman markers to determine separate thermodynamic contributions of A, T and deoxyribosyl-phosphate moieties to premelting and melting transitions. Key findings include the following: (i) Both poly(dA)·poly(dT) and poly(dA–dT)· poly(dA–dT) exhibit robust premelting transitions, due predominantly to backbone conformational changes. (ii) The significant van’t Hoff premelting enthalpies of poly(dA)·poly(dT) [ΔH_vH^pm = 18.0 ± 1.6 kcal·mol^–1 (kilocalories per mole cooperative unit)] and poly(dA–dT)·poly(dA–dT) (ΔH_vH^pm = 13.4 ± 2.5 kcal·mol^–1) differ by an amount (~4.6 kcal·mol^–1) estimated as the contribution from three-centered inter-base hydrogen bonding in (dA)_n·(dT)_n tracts. (iii) The overall stacking free energy of poly(dA)· poly(dT) [–6.88 kcal·mol_bp^–1 (kilocalories per mole base pair)] is greater than that of poly(dA–dT)· poly(dA–dT) (–6.31 kcal·mol_bp^–1). (iv) The difference between stacking free energies of A and T is significant in poly(dA)·poly(dT) (ΔΔG_st = 0.8 ± 0.3 kcal· mol_bp^–1), but marginal in poly(dA–dT)·poly(dA–dT) (ΔΔG_st = 0.3 ± 0.3 kcal·mol_bp^–1). (v) In poly(dA)· poly(dT), the van’t Hoff parameters for melting of A (ΔH_vH^A = 407 ± 23 kcal·mol^–1, ΔS_vH^A = 1166 ± 67 cal·°K^–1·mol^–1, ΔG_vH(25°C)^A = 60.0 ± 3.2 kcal·mol^–1) are clearly distinguished from those of T (ΔH_vH^T = 185 ± 38 kcal·mol^–1, ΔS_vH^T = 516 ± 109 cal·°K^–1·mol^–1, ΔG_vH(25°C)^T = 27.1 ± 5.5 kcal·mol^–1). (vi) Similar relative differences are observed in poly(dA–dT)· poly(dA–dT) (ΔH_vH^A = 333 ± 54 kcal·mol^–1, ΔS_vH^A = 961 ± 157 cal·°K^–1·mol^–1, ΔG_vH(25°C)^A = 45.0 ± 7.6 kcal· mol^–1; ΔH_vH^T = 213 ± 30 kcal·mol^–1, ΔS_vH^T = 617 ± 86 cal·°K^–1·mol^–1, ΔG_vH(25°C)^T = 29.3 ± 4.9 kcal·mol^–1). The methodology employed here distinguishes thermodynamic contributions of base stacking, base pairing and backbone conformational ordering in the molecular mechanism of double-helical B DNA formation.     

Malfunctioning of the Iron–Sulfur Cluster Assembly Machinery in Saccharomyces cerevisiae Produces Oxidative Stress via an Iron-Dependent Mechanism,Causing Dysfunction in Respiratory Complexes

Biogenesis and recycling of iron–sulfur (Fe–S) clusters play important roles in the iron homeostasis mechanisms involved in mitochondrial function. In Saccharomyces cerevisiae, the Fe–S clusters are assembled into apoproteins by the iron–sulfur cluster machinery (ISC). The aim of the present study was to determine the effects of ISC gene deletion and consequent iron release under oxidative stress conditions on mitochondrial functionality in S. cerevisiae. Reactive oxygen species (ROS) generation, caused by H₂O₂, menadione, or ethanol, was associated with a loss of iron homeostasis and exacerbated by ISC system dysfunction. ISC mutants showed increased free Fe²⁺ content, exacerbated by ROS-inducers, causing an increase in ROS, which was decreased by the addition of an iron chelator. Our study suggests that the increment in free Fe²⁺ associated with ROS generation may have originated from mitochondria, probably Fe–S cluster proteins, under both normal and oxidative stress conditions, suggesting that Fe–S cluster anabolism is affected. Raman spectroscopy analysis and immunoblotting indicated that in mitochondria from SSQ1 and ISA1 mutants, the content of [Fe–S] centers was decreased, as was formation of Rieske protein-dependent supercomplex III₂IV₂, but this was not observed in the iron-deficient ATX1 and MRS4 mutants. In addition, the activity of complexes II and IV from the electron transport chain (ETC) was impaired or totally abolished in SSQ1 and ISA1 mutants. These results confirm that the ISC system plays important roles in iron homeostasis, ROS stress, and in assembly of supercomplexes III₂IV₂ and III₂IV₁, thus affecting the functionality of the respiratory chain.     

Synthesis,in vitro stability,and antiproliferative effect of d‐cysteine modified GnRH‐doxorubicin conjugates

Overexpression of gonadotropin‐releasing hormone (GnRH) receptor in many tumors but not in normal tissues makes it possible to use GnRH analogs as targeting peptides for selective delivery of cytotoxic agents, which may help to enhance the uptake of anticancer drugs by cancer cells and reduce toxicity to normal cells. The GnRH analogs [d ‐Cys⁶, desGly¹⁰, Pro⁹‐NH₂]‐GnRH, [d ‐Cys⁶, desGly¹⁰, Pro⁹‐NHEt]‐GnRH, and [d ‐Cys⁶, α‐aza‐Gly¹⁰‐NH₂]‐GnRH were conjugated with doxorubicin (Dox), respectively, through N‐succinimidyl‐3‐maleimidopropionate as a linker to afford three new GnRH‐Dox conjugates. The metabolic stability of these conjugates in human serum was determined by RP‐HPLC. The antiproliferative activity of the conjugates was examined in GnRH receptor‐positive MCF‐7 human breast cancer cell line by MTT assay. The three GnRH‐Dox conjugates showed improved metabolic stability in human serum in comparison with AN‐152. The antiproliferative effect of conjugate II ([d ‐Cys⁶, desGly¹⁰, Pro⁹‐NHEt]‐GnRH‐Dox) on MCF‐7 cells was higher than that of conjugate I ([d ‐Cys⁶, desGly¹⁰, Pro⁹‐NH₂]‐GnRH‐Dox) and conjugate III ([d ‐Cys⁶, α‐aza‐Gly¹⁰‐NH₂]‐GnRH‐Dox), and the cytotoxicity of conjugate II against GnRH receptor‐negative 3T3 mouse embryo fibroblast cells was decreased in comparison with free Dox. GnRH receptor inhibition test suggested that the antiproliferative activity of conjugate II might be due to the cellular uptake mediated by the targeting binding of [d ‐Cys⁶‐des‐Gly¹⁰‐Pro⁹‐NHEt]‐GnRH to GnRH receptors. Our study indicates that targeting delivery of conjugate II mediated by [d ‐Cys⁶‐des‐Gly¹⁰‐Pro⁹‐NHEt]‐GnRH is a promising strategy for chemotherapy of tumors that overexpress GnRH receptors.     

Crystal structure of parallel G-quadruplex formed by the two-repeat ALS- and FTD-related GGGGCC sequence

The hexanucleotide repeat expansion, GGGGCC (G4C2), within the first intron of the C9orf72 gene is known to be the most common genetic cause of both amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The G4C2 repeat expansions, either DNA or RNA, are able to form G-quadruplexes which induce toxicity leading to ALS/FTD. Herein, we report a novel crystal structure of d(G4C2)₂ that self-associates to form an eight-layer parallel tetrameric G-quadruplex. Two d(G4C2)₂ associate together as a parallel dimeric G-quadruplex which folds into a tetramer via 5′-to-5′ arrangements. Each dimer consists of four G-tetrads connected by two CC propeller loops. Especially, the 3′-end cytosines protrude out and form C·C+•C·C+/ C·C•C·C+ quadruple base pair or C•C·C+ triple base pair stacking on the dimeric block. Our work sheds light on the G-quadruplexes adopted by d(G4C2) and yields the invaluable structural details for the development of small molecules to tackle neurodegenerative diseases, ALS and FTD.     

Requirements of the cytosolic iron–sulfur cluster assembly pathway in Arabidopsis

The assembly of iron–sulfur (Fe–S) clusters requires dedicated protein factors inside the living cell. Striking similarities between prokaryotic and eukaryotic assembly proteins suggest that plant cells inherited two different pathways through endosymbiosis: the ISC pathway in mitochondria and the SUF pathway in plastids. Fe–S proteins are also found in the cytosol and nucleus, but little is known about how they are assembled in plant cells. Here, we show that neither plastid assembly proteins nor the cytosolic cysteine desulfurase ABA3 are required for the activity of cytosolic aconitase, which depends on a [4Fe–4S] cluster. In contrast, cytosolic aconitase activity depended on the mitochondrial cysteine desulfurase NFS1 and the mitochondrial transporter ATM3. In addition, we were able to complement a yeast mutant in the cytosolic Fe–S cluster assembly pathway, dre2, with the Arabidopsis homologue AtDRE2, but only when expressed together with the diflavin reductase AtTAH18. Spectroscopic characterization showed that purified AtDRE2 could bind up to two Fe–S clusters. Purified AtTAH18 bound one flavin per molecule and was able to accept electrons from NAD(P)H. These results suggest that the proteins involved in cytosolic Fe–S cluster assembly are highly conserved, and that dependence on the mitochondria arose before the second endosymbiosis event leading to plastids.     

Production of Italian Dry Salami: Effect of Starter Culture and Chemical Acidulation on Staphylococcal Growth in Salami Under Commercial Manufacturing Conditions

The effect of starter culture and chemical acidulation on the growth and enterotoxigenesis of Staphylococcus aureus strain S-6 in Italian dry salami under commercial manufacturing conditions was studied. The experimental design included two levels of S. aureus (10⁴ and 10⁵/g), three levels of starter culture (0, 10⁵, and 10⁶/g), three levels of initial pH (pH₀) (6.1, 5.5, and 4.8), two manufacturing plants, and three replications. S. aureus growth in the salami was affected significantly (P < 0.005) by pH₀, initial levels of S. aureus (staph₀) and lactic acid bacteria (LAB₀), day of fermentation, and by the interactions of pH₀ × day, pH₀ × LAB₀, LAB₀ × staph₀, pH₀ × staph₀, and pH₀ × location of fermentation. In general, the lower the pH₀ and the higher the LAB₀, the greater the inhibition of S. aureus. The LAB levels during the fermentation were affected significantly (P < 0.005) by pH₀, LAB₀, day of fermentation, location, LAB₀ × pH₀, and LAB₀ × day. Derived regression equations related level of S. aureus and LAB at any day of fermentation to a number of microbiological and chemical variables. Close similarity of observed and predicted levels of S. aureus and LAB growth demonstrated the usefulness of the experimental approach in evaluating the safety of a process. No detectable enterotoxin or thermonuclease was found at any stage of processing even when S. aureus reached levels of 10⁷/g of salami.     

The function of glutaredoxin GRXS15 is required for lipoyl-dependent dehydrogenases in mitochondria

Iron–sulfur (Fe–S) clusters are ubiquitous cofactors in all life and are used in a wide array of diverse biological processes, including electron transfer chains and several metabolic pathways. Biosynthesis machineries for Fe–S clusters exist in plastids, the cytosol, and mitochondria. A single monothiol glutaredoxin (GRX) is involved in Fe–S cluster assembly in mitochondria of yeast and mammals. In plants, the role of the mitochondrial homolog GRXS15 has only partially been characterized. Arabidopsis (Arabidopsis thaliana) grxs15 null mutants are not viable, but mutants complemented with the variant GRXS15 K83A develop with a dwarf phenotype similar to the knockdown line GRXS15^amiR. In an in-depth metabolic analysis of the variant and knockdown GRXS15 lines, we show that most Fe–S cluster-dependent processes are not affected, including biotin biosynthesis, molybdenum cofactor biosynthesis, the electron transport chain, and aconitase in the tricarboxylic acid (TCA) cycle. Instead, we observed an increase in most TCA cycle intermediates and amino acids, especially pyruvate, glycine, and branched-chain amino acids (BCAAs). Additionally, we found an accumulation of branched-chain α-keto acids (BCKAs), the first degradation products resulting from transamination of BCAAs. In wild-type plants, pyruvate, glycine, and BCKAs are all metabolized through decarboxylation by mitochondrial lipoyl cofactor (LC)-dependent dehydrogenase complexes. These enzyme complexes are very abundant, comprising a major sink for LC. Because biosynthesis of LC depends on continuous Fe–S cluster supply to lipoyl synthase, this could explain why LC-dependent processes are most sensitive to restricted Fe–S supply in grxs15 mutants.     

Structural characterization of cationic lipid–tRNA complexes

Despite considerable interest and investigations on cationic lipid–DNA complexes, reports on lipid–RNA interaction are very limited. In contrast to lipid–DNA complexes where lipid binding induces partial B to A and B to C conformational changes, lipid–tRNA complexation preserves tRNA folded state. This study is the first attempt to investigate the binding of cationic lipid with transfer RNA and the effect of lipid complexation on tRNA aggregation and condensation. We examine the interaction of tRNA with cholesterol (Chol), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), dioctadecyldimethylammoniumbromide (DDAB) and dioleoylphosphatidylethanolamine (DOPE), at physiological condition, using constant tRNA concentration and various lipid contents. FTIR, UV-visible, CD spectroscopic methods and atomic force microscopy (AFM) were used to analyze lipid binding site, the binding constant and the effects of lipid interaction on tRNA stability, conformation and condensation. Structural analysis showed lipid–tRNA interactions with G–C and A–U base pairs as well as the backbone phosphate group with overall binding constants of K_Chol = 5.94 (± 0.8) × 10⁴ M^–1, K_DDAB = 8.33 (± 0.90) × 10⁵ M^–1, K_DOTAP = 1.05 (± 0.30) × 10⁵ M^–1 and K_DOPE = 2.75 (± 0.50) × 10⁴ M^–1. The order of stability of lipid–tRNA complexation is DDAB > DOTAP > Chol > DOPE. Hydrophobic interactions between lipid aliphatic tails and tRNA were observed. RNA remains in A-family structure, while biopolymer aggregation and condensation occurred at high lipid concentrations.     

Phosphoenolpyruvate Carboxykinase Is Involved in the Decarboxylation of Aspartate in the Bundle Sheath of Maize

We recently showed that maize (Zea mays L.) leaves contain appreciable amounts of phosphoenolpyruvate carboxykinase (PEPCK; R.P. Walker, R.M. Acheson, L.I. Técsi, R.C. Leegood [1997] Aust J Plant Physiol 24: 459–468). In the present study, we investigated the role of PEPCK in C₄ photosynthesis in maize. PEPCK activity and protein were enriched in extracts from bundle-sheath (BS) strands compared with whole-leaf extracts. Decarboxylation of [4-¹⁴C]aspartate (Asp) by BS strands was dependent on the presence of 2-oxoglutarate and Mn²⁺, was stimulated by ATP, was inhibited by the PEPCK-specific inhibitor 3-mercaptopicolinic acid, and was independent of illumination. The principal product of Asp metabolism was phosphoenolpyruvate, whereas pyruvate was a minor product. Decarboxylation of [4-¹⁴C]malate was stimulated severalfold by Asp and 3-phosphoglycerate, was only slightly reduced in the absence of Mn²⁺ or in the presence of 3-mercaptopicolinic acid, and was light dependent. Our data show that decarboxylation of Asp and malate in BS cells of maize occurs via two different pathways: Whereas malate is mainly decarboxylated by NADP-malic enzyme, decarboxylation of Asp is dependent on the activity of PEPCK.     

Structural analysis of DNA complexation with cationic lipids

Complexes of cationic liposomes with DNA are promising tools to deliver genetic information into cells for gene therapy and vaccines. Electrostatic interaction is thought to be the major force in lipid–DNA interaction, while lipid-base binding and the stability of cationic lipid–DNA complexes have been the subject of more debate in recent years. The aim of this study was to examine the complexation of calf-thymus DNA with cholesterol (Chol), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), dioctadecyldimethylammoniumbromide (DDAB) and dioleoylphosphatidylethanolamine (DOPE), at physiological condition, using constant DNA concentration and various lipid contents. Fourier transform infrared (FTIR), UV-visible, circular dichroism spectroscopic methods and atomic force microscopy were used to analyse lipid-binding site, the binding constant and the effects of lipid interaction on DNA stability and conformation. Structural analysis showed a strong lipid–DNA interaction via major and minor grooves and the backbone phosphate group with overall binding constants of K_Chol = 1.4 (±0.5) × 10⁴ M⁻¹, K_DDAB = 2.4 (±0.80) × 10⁴ M⁻¹, K_DOTAP = 3.1 (±0.90) × 10⁴ M⁻¹ and K_DOPE = 1.45 (± 0.60) × 10⁴ M⁻¹. The order of stability of lipid–DNA complexation is DOTAP>DDAB>DOPE>Chol. Hydrophobic interactions between lipid aliphatic tails and DNA were observed. Chol and DOPE induced a partial B to A-DNA conformational transition, while a partial B to C-DNA alteration occurred for DDAB and DOTAP at high lipid concentrations. DNA aggregation was observed at high lipid content.     

Mutational analyses of dinucleotide and tetranucleotide microsatellites in Escherichia coli: influence of sequence on expansion mutagenesis

Mutagenesis at [GT/CA]₁₀, [TC/AG]₁₁ and [TTCC/AAGG]₉ microsatellite sequences inserted in the herpes simplex virus thymidine kinase (HSV-tk) gene was analyzed in isogenic mutL⁺ and mutL^– Escherichia coli. In both strains, significantly more expansion than deletion mutations were observed at the [TTCC/AAGG]₉ motif relative to either dinucleo­tide motif. As the HSV-tk coding sequence contains an endogenous [G/C]₇ mononucleotide repeat and ~1000 bp of unique sequence, we were able to compare mutagenesis among various sequence motifs. We observed that the relative risk of mutation in E.coli is: [TTCC/AAGG]₉ > [GT/CA]₁₀ ~ [TC/AG]₁₁ > unique ~ [G/C]₇. The mutation frequency varied 1400-fold in mutL⁺ cells between the tetranucleotide motif and the mononucleotide motif, but only 50-fold in mutL^– cells. The [G/C]₇ sequence was destabilized the greatest and the tetranucleotide motif the least by loss of mismatch repair. These results demonstrate that the quantitative risk of mutation at various microsatellites greatly depends on the DNA sequence composition. We suggest alternative models for the production of expansion mutations during lagging strand replication of the [TTCC/AAGG]₉ microsatellite.     

A novel replicating circular DNAzyme

10–23 DNAzyme has the potential to suppress gene expressions through sequence-specific mRNA cleavage. However, the dependence on exogenous delivery limits its applications. The objective of this work is to establish a replicating DNAzyme in bacteria using a single-stranded DNA vector. By cloning the 10–23 DNAzyme into the M13mp18 vector, we constructed two circular DNAzymes, C-Dz₇ and C-Dz₄₈₂, targeting the β-lactamase mRNA. These circular DNAzymes showed in vitro catalytic efficiencies (k_cat/K_M) of 7.82 × 10⁶ and 1.36 × 10⁷ M^–1·min^–1, respectively. Their dependence on divalent metal ions is similar to that found with linear 10–23 DNAzyme. Importantly, the circular DNAzymes were not only capable of replicating in bacteria but also exhibited high activities in inhibiting β-lactamase and bacterial growth. This study thus provides a novel strategy to produce replicating DNAzymes which may find widespread applications.     

Biosynthesis of the sactipeptide Ruminococcin C by the human microbiome: Mechanistic insights into thioether bond formation by radical SAM enzymes

Despite its major importance in human health, the metabolic potential of the human gut microbiota is still poorly understood. We have recently shown that biosynthesis of Ruminococcin C (RumC), a novel ribosomally synthesized and posttranslationally modified peptide (RiPP) produced by the commensal bacterium Ruminococcus gnavus, requires two radical SAM enzymes (RumMC1 and RumMC2) catalyzing the formation of four C_α-thioether bridges. These bridges, which are essential for RumC''s antibiotic properties against human pathogens such as Clostridium perfringens, define two hairpin domains giving this sactipeptide (sulfur-to-α-carbon thioether–containing peptide) an unusual architecture among natural products. We report here the biochemical and spectroscopic characterizations of RumMC2. EPR spectroscopy and mutagenesis data support that RumMC2 is a member of the large family of SPASM domain radical SAM enzymes characterized by the presence of three [4Fe-4S] clusters. We also demonstrate that this enzyme initiates its reaction by C_α H-atom abstraction and is able to catalyze the formation of nonnatural thioether bonds in engineered peptide substrates. Unexpectedly, our data support the formation of a ketoimine rather than an α,β-dehydro-amino acid intermediate during C_α-thioether bridge LC–MS/MS fragmentation. Finally, we explored the roles of the leader peptide and of the RiPP precursor peptide recognition element, present in myriad RiPP-modifying enzymes. Collectively, our data support a more complex role for the peptide recognition element and the core peptide for the installation of posttranslational modifications in RiPPs than previously anticipated and suggest a possible reaction intermediate for thioether bond formation.     

Efficiency and pattern of UV pulse laser-induced RNA-RNA cross-linking in the ribosome

Escherichia coli ribosomes were irradiated with a KrF excimer laser (248 nm, 22 ns pulse) with incident pulse energies in the range of 10–40 mJ for a 1 cm² area, corresponding to fluences of 4.5 to 18 × 10⁹ W m^–2, to determine strand breakage yields and the frequency and pattern of RNA–RNA cross- linking in the 16S rRNA. Samples were irradiated in a cuvette with one laser pulse or in a flow cell with an average of 4.6 pulses per sample. The yield of strand breaks per photon was intensity dependent, with values of 0.7 to 1.3 × 10^–3 over the incident intensity range studied. The yield for RNA–RNA cross-linking was 3 × 10^–4 cross-links/photon at the intensity of 4.5 × 10⁹ W m^–2, an ~4-fold higher yield per photon than obtained with a transilluminator. The cross-link yield/photon decreased at higher light intensities, probably due to intensity-dependent photoreversal. The pattern of cross-linking was similar to that observed with low intensity irradiation but with four additional long-range cross-links not previously seen in E.coli ribosomes. Cross- linking frequencies obtained with one laser pulse are more correlated to internucleotide distances than are frequencies obtained with transilluminator irradiation.     

Erythrocruorin of Marphysa sanguinea. Isolation of some physical, physicochemical and other properties

1. The polychaete worm Marphysa sanguinea has a circulating erythrocruorin of mol.wt. about 2·4×10⁶ (S⁰_20,w 58·2s, D_20,w 2·06×10⁻⁷ cm.²/sec). This is the predominant form existing at pH 6–8 and (non-protein) I 0·10–0·21, and also at approx. pH 6·7 and I 0·15–3·00. 2. The pigment contains 2·24% of protohaem. 3. The 58s protein has an electrophoretic mobility of 8·08×10⁻⁵ cm.²/v/sec. at pH 8·12, I 0·21 and 0°. The isoelectric point of suspended particles is 4·63 at I 0·16 and 21·5°. 4. At very low ionic strength and pH 6·7 (unbuffered) the 58s pigment associates reversibly to 97s and 150s forms, which are probably dimer and tetramer species. 5. At pH 10·0 and I 0·025, it dissociates irreversibly to give a small amount of 2–4s non-haem-containing protein and much 9s haem-enriched protein. These and the 58s pigment may correspond to structures found in Levin's (1963) electron-microscope studies of other erythrocruorins. 6. Absorption spectra of the 58s oxygenated erythrocruorin and the deoxygenated and carbon monoxide derivatives have been obtained.     

Carex jianfengensis (Carex sect. Rhomboidales,Cyperaceae), a New Species from Hainan,China

A new species of Carex sect. Rhomboidales, C. jianfengensis, is described and illustrated from Hainan, China. The new species is similar to C. zunyiensis but differs in having involucral bracts sparsely hispid and with ca.1 cm long sheaths; inflorescence with 4 spikes, terminal spike ca. 2.5 cm long, lateral spikes 2–3.5 × 0.7–1 cm; staminate glumes narrowly ovate, ca. 5 mm; pistillate glumes triangular-lanceolate, 5–7 mm; perigynia 6–8 × 3 mm and pubescent on veins; nutlet 4–5 mm long, rhombic-ovoid, trigonous, base with shortly stipitate, apex abruptly contracted into a erect short beak, and not expanding into an annulate orifice.     

Structural and Molecular Basis of the Peroxynitrite-mediated Nitration and Inactivation of Trypanosoma cruzi Iron-Superoxide Dismutases (Fe-SODs) A and B: DISPARATE SUSCEPTIBILITIES DUE TO THE REPAIR OF TYR35 RADICAL BY CYS83 IN Fe-SODB THROUGH INTRAMOLECULAR ELECTRON TRANSFER*

Trypanosoma cruzi, the causative agent of Chagas disease, contains exclusively iron-dependent superoxide dismutases (Fe-SODs) located in different subcellular compartments. Peroxynitrite, a key cytotoxic and oxidizing effector biomolecule, reacted with T. cruzi mitochondrial (Fe-SODA) and cytosolic (Fe-SODB) SODs with second order rate constants of 4.6 ± 0.2 × 10⁴ m⁻¹ s⁻¹ and 4.3 ± 0.4 × 10⁴ m⁻¹ s⁻¹ at pH 7.4 and 37 °C, respectively. Both isoforms are dose-dependently nitrated and inactivated by peroxynitrite. Susceptibility of T. cruzi Fe-SODA toward peroxynitrite was similar to that reported previously for Escherichia coli Mn- and Fe-SODs and mammalian Mn-SOD, whereas Fe-SODB was exceptionally resistant to oxidant-mediated inactivation. We report mass spectrometry analysis indicating that peroxynitrite-mediated inactivation of T. cruzi Fe-SODs is due to the site-specific nitration of the critical and universally conserved Tyr³⁵. Searching for structural differences, the crystal structure of Fe-SODA was solved at 2.2 Å resolution. Structural analysis comparing both Fe-SOD isoforms reveals differences in key cysteines and tryptophan residues. Thiol alkylation of Fe-SODB cysteines made the enzyme more susceptible to peroxynitrite. In particular, Cys⁸³ mutation (C83S, absent in Fe-SODA) increased the Fe-SODB sensitivity toward peroxynitrite. Molecular dynamics, electron paramagnetic resonance, and immunospin trapping analysis revealed that Cys⁸³ present in Fe-SODB acts as an electron donor that repairs Tyr³⁵ radical via intramolecular electron transfer, preventing peroxynitrite-dependent nitration and consequent inactivation of Fe-SODB. Parasites exposed to exogenous or endogenous sources of peroxynitrite resulted in nitration and inactivation of Fe-SODA but not Fe-SODB, suggesting that these enzymes play distinctive biological roles during parasite infection of mammalian cells.