The nine-membered indolactam antibiotics belong to a small group of antibiotics showing broad biological activities. However, the in vivo genetic engineering of compounds of this type has not been performed. Here we report the identification of a single gene cluster responsible for the biosynthesis of methylpendolmycin and pendolmycin, two members of this family of antibiotics, from the deep sea bacterium Marinactinospora thermotolerans SCSIO 00652. Bioinformatics analysis and gene inactivation, coupled with metabolite characterization, reveal that MpnB, a nonribosomal peptide synthetase, MpnC, a cytochrome P450, and MpnD, a prenyltransferase, are sufficient to catalyze the biosynthesis of the two antibiotics from L-Ile (or L-Val), L-Trp, and methionine. MpnD is the first identified enzyme that transfers a C5 prenyl unit in a reverse manner to the C-7 position of a Trp-derived natural product.

Indolactam alkaloids: A single gene cluster responsible for biosynthesis of methylpendolmycin and pendolmycin has been characterized. Bioinformatics analysis and gene inactivation, coupled with metabolite characterization, reveal that MpnB (a nonribosomal peptide synthetase), MpnC (a cytochrome P450), and MpnD (a prenyltransferase) are sufficient to catalyze the biosynthesis of the two antibiotics from L-Ile (or L-Val), L-Trp, and methionine.

The O-acetylation of peptidoglycan (PG) is now known to occur in 53 species, including numerous human pathogens such as, Staphylococcus aureus, Bacillus anthracis, species of Enterococcus, Campylobacter jejuni, Helicobacter pylori, Neisseria gonorrhoeae and N. meningitidis. This modification, which occurs at the C-6 hydroxyl of N-acetylmuramoyl residues within PG, serves to regulate autolytic activity during PG metabolism and contributes to pathogenesis and persistence within a host. O-Acetylpeptidoglycan esterase (Ape) was recently discovered as an enzyme responsible for the removal of O-acetyl groups from PG, thus permitting the continued maintenance and metabolism of the sacculus. Recombinant Ape1 from N. gonorrhoeae was purified to apparent homogeneity and optimal storage conditions for the enzyme were determined. Using 4-methylumbelliferyl acetate as substrate, a fluorogenic assay amenable for the high-throughput screening for potential inhibitors was developed and Ape1 was screened against a subset of compounds of the Canadian Compound Collection. The overall Z′ score for the screen was 0.62, indicative of a well-suited assay with a sufficient signal window, and the threshold was set at 65 %. After eliminating a number of false-positives, seven compounds were identified as true inhibitors of Ape1, the first to be identified for this class of enzyme. Dose–response curves were generated leading to the identification of five of these compounds with IC₅₀ values ranging between 0.3 and 23 μM. Of these, purpurin was selected for further analysis and it was found to inhibit the growth of both Gram-positive and Gram-negative bacteria that produce both O-acetylated PG and Ape.

A high-throughput screen was developed to identify the first inhibitors of O-acetylpeptidoglycan esterase, the enzyme responsible for the removal of the O-acetyl groups of the peptidoglycan of many pathogenic bacteria. Seven compounds differing in structure were found and further analysis validated the enzyme as a potential new antibacterial target.

Protein modification with isoprenoid lipids affects hundreds of signaling proteins in eukaryotic cells. Modification of isoprenoids with reporter groups is the main approach for the creation of probes for the analysis of protein prenylation in vitro and in vivo. Here, we describe a new strategy for the synthesis of functionalized phosphoisoprenoids that uses an aminederivatized isoprenoid scaffold as a starting point for the synthesis of functionalized phosphoisoprenoid libraries. This overcomes a long-standing problem in the field, where multistep synthesis had to be carried out for each individual isoprenoid analogue. The described approach enabled us to synthesize a range of new compounds, including two novel fluorescent isoprenoids that previously could not be generated by conventional means. The fluorescent probes that were developed using the described approach possess significant spectroscopic advantages to all previously generated fluorescent isoprenoid analogue. Using these analogues for flow cytometry and cell imaging, we analyzed the uptake of isoprenoids by mammalian cells and zebrafish embryos. Furthermore, we demonstrate that derivatization of the scaffold can be coupled in a one-pot reaction to enzymatic incorporation of the resulting isoprenoid group into proteins. This enables rapid evaluation of functional groups for compatibility with individual prenyltransferases and identification of the prenyltransferase specific substrates.

Prenylation probes: Eukaryotic protein prenyltransferases modify polypeptides with isoprenoid lipids. Modification of isoprenoids with reporter groups allows the creation of probes for the analysis of protein prenylation in vitro and in vivo. An amine-derivatized isoprenoid scaffold was used as a novel starting point for the synthesis of functionalized phosphoisoprenoid libraries.

The His-Asn-His (HNH) motif characterizes the active sites of a large number of different nucleases such as homing endonucleases, restriction endonucleases, structure-specific nucleases and, in particular, nonspecific nucleases. Several biochemical studies have revealed an essential catalytic function for the first amino acid of this motif in HNH nucleases. This histidine residue was identified as the general base that activates a water molecule for a nucleophilic attack on the sugar phosphate backbone of nucleic acids. Replacement of histidine by an amino acid such as glycine or alanine, which lack the catalytically active imidazole side chain, leads to decreases of several orders of magnitude in the nucleolytic activities of members of this nuclease family. We were able, however, to restore the activity of HNH nuclease variants (i.e., EndA (Streptococcus pneumoniae), SmaNuc (Serratia marcescens) and NucA (Anabaena sp.)) that had been inactivated by His→Gly or His→Ala substitution by adding excess imidazole to the inactive enzymes in vitro. Imidazole clearly replaces the missing histidine side chain and thereby restores nucleolytic activity. Significantly, this chemical rescue could also be observed in vivo (Escherichia coli). The in vivo assay might be a promising starting point for the development of a high-throughput screening system for functional EndA inhibitors because, unlike the wild-type enzyme, the H160G and H160A variants of EndA can easily be produced in E. coli. A simple viability assay would allow inhibitors of EndA to be identified because these would counteract the toxicities of the chemically rescued EndA variants. Such inhibitors could be used to block the nucleolytic activity of EndA, which as a surface-exposed enzyme in its natural host destroys the DNA scaffolds of neutrophil extracellular traps (NETs) and thereby allows S. pneumoniae to escape the innate immune response.

Nuclease activation: The HNH motif characterizes many nucleases, among them EndA from Streptococcus pneumoniae. One of the His residues is responsible for activating a water molecule for phosphodiester bond cleavage. Its replacement by other amino acids leads to inactive enzymes. However, subsequent addition of imidazole can rescue catalytic activity in vitro and in vivo.

Seven-arm chaperone: A modest μM inhibitor of glucosylceramide β-glucosidase (GCase) has been transformed into a potent low-nM inhibitor by multivalency. This iminosugar inhibitor acts as a pharmacological chaperone and increases residual GCase activity in fibroblasts from Gaucher patients. These results open the way to a new class of chaperones for the treatment of lysosomal diseases.

We describe a novel method for the isolation of selective protease variants displayed on the surface of E. coli. The method relies on the electrostatic capture of an autoinhibited protein on the cell surface, combined with external labeling using a fluorophore-conjugated binding peptide (PMI-FL). Using this method, we isolated an OmpT variant that hydrolyzes a target sequence (between Ala-Arg) with high selectivity.

Snapshots of spliceosome assembly: A high-throughput in vitro splicing assay has been developed for screening a compound library. The discovered family of pre-mRNA splicing inhibitors comprises lichen secondary metabolites that allow the enrichment of the spliceosomal complexes formed after the spliceosome activation step. Here we identify the structural features important for the compounds' inhibitory activity.

The ubiquitously expressed Abl protein is a non-receptor tyrosine kinase that undergoes nuclear–cytoplasmic shuttling and is involved in many signaling pathways in the cell. Nuclear Abl is activated by DNA damage to regulate DNA repair, cell-cycle checkpoints and apoptosis. Previous studies have established that ataxia telangiectasia mutated (ATM) activates nuclear Abl by phosphorylating serine 465 (S465) in the kinase domain in response to ionizing radiation (IR). Using a peptide biosensor that specifically reports on the Abl kinase activity, we found that an Abl-S465A mutant, which is not capable of being activated by ATM through the canonical site, was still activated rapidly after IR. We established that DNA-dependent protein kinase (DNAPK) is likely to be responsible for a second pathway to activate Abl early on in the response to IR through phosphorylation at a site other than S465. Our findings show that nuclear and cytoplasmic Abl kinase is activated early on (within 5 min) in response to IR by both ATM and DNAPK, and that although one or the other of these kinases is required, either one is sufficient to activate Abl. These results support the concept of early Abl recruitment by both the ATM and the DNAPK pathways to regulate nuclear events triggered by DNA damage and potentially communicate them to proteins in the cytoplasm.

Damage limitation: A peptide biosensor for intracellular Abl kinase activation helps elucidate a potential, early, ATM- and DNAPK-dependent role for Abl in the response to DNA damage from ionizing radiation. The substrate peptide is combined with a binding sequence and a cell-permeability tag to enable uptake and specific phosphorylation by overexpressed Abl kinase in the cell.

Organophosphonates are widespread in nature, and their catabolic pathways are of great importance for many microorganisms. In a remarkable series of biochemical experiments, Raushel and co-workers reconstituted the carbon–phosphorus lyase activity, which cleaves the CP bond of 5-phospho-α-D-ribosyl-methylphosphonate to produce methane and a cyclic phosphodiester in a reaction that requires S-adenosylmethionine and a [4Fe–4S] cluster.

Two-pronged attack: We describe the maturation of a bivalent aptamer by a chemically driven two-step process. From an improved monovalent aptamer subdomain that had been modified by polycyclic aromatic hydrocarbons at individual positions, a mature bivalent variant with superior activities to its progenitor molecule was obtained through domain reassembly.

The influence of water: Crystallization of (R/S)-α,β-CHF-dATP with the preorganized pol β-DNA complex shows that (S)-α,β-CHF-dATP is preferentially bound to the active site with the CF fluorine proximal to a structural water bound to Asp276.

On the tracks of a DNA polymerase: NMR provides insights into DNA synthesis in a virtually label-free manner and under close-to-physiological conditions. Through the monitoring of the chemical-shift changes of multiple ¹³C-methyl methionine residues we found unique recognition states for canonical and noncanonical cases, thus indicating enzymatic cycling through distinct paths.

Hold your P's: Phosphorylated tyrosine and serine residues in peptides have been modified selectively by DNA catalysts (see graphic). These deoxyribozymes catalyze covalent attachment of an RNA tag to a range of peptide sequences, thus a proof of principle for a new approach to phosphopeptide analysis is established.

Two pyrene-modified UNA monomers were synthesized and incorporated into 21-mer DNA oligonucleotides. Melting temperatures and thermodynamic properties of the modified duplexes were measured, and the fluorescence properties of single strands and duplexes containing one or more pyrene-UNA modifications were studied. It was found that incorporation of pyrene-UNA monomers increased duplex stability relative to UNA monomers, and thermodynamic studies revealed significant mismatch discriminative capabilities of the pyrene-UNA modified oligonucleotides. Furthermore, the steady-state fluorescence emission intensities of pyrene-UNA modified oligonucleotides were increased upon hybridization to DNA, which to the best of our knowledge is unprecedented for an acyclic pyrene modification in DNA. Interestingly, pyrene excimer emission was observed for single-stranded oligonucleotides containing three pyrene-UNA modifications, whereas this excimer emission disappeared after hybridization to DNA. In view of both the pyrene monomer and the excimer fluorescence emission, the triply modified oligonucleotides show intriguing properties relating to the development of new DNA/RNA detection tools.

We have combined the fluorescent properties of pyrene with the acyclic UNA (unlocked nucleic acid) scaffold to produce novel fluorescent oligonucleotides with high mismatch discriminative power and the ability to detect DNA targets in homogeneous fluorescence assays. These probes therefore show great promise in the field of nucleic acid diagnostics.

MicroRNAs (miRNAs) have crucial functions in many cellular processes, such as differentiation, proliferation and apoptosis; aberrant expression of miRNAs has been linked to human diseases, including cancer. Tools that allow specific and efficient knockdown of miRNAs would be of immense importance for exploring miRNA function. Zebrafish serves as an excellent vertebrate model system to understand the functions of miRNAs involved in a variety of biological processes. We designed and employed a strategy based on locked nucleic acid enzymes (LNAzymes) for in vivo knockdown of miRNA in zebrafish embryos. We demonstrate that LNAzyme can efficiently knockdown miRNAs with minimal toxicity to the zebrafish embryos.

In vivo miRNA assassination: We designed and employed a strategy with locked nucleic acid enzyme (LNAzyme) for in vivo knockdown of microRNA (miRNA) in zebrafish embryos. We demonstrate that LNAzyme can efficiently knockdown miRNAs with minimal toxicity to the zebrafish embryos.

Elucidation of the binding modes of Ty inhibitors is an important step for in-depth studies on how to regulate tyrosinase activity. In this paper we highlight the extraordinarily versatile effects of the aurone structure on mushroom Ty activity. Depending on the position of the OH group on the B-ring, aurones can behave either as substrates or as hyperbolic activators. The synthesis of a hybrid aurone through combination of an aurone moiety with HOPNO (2-hydroxypyridine N-oxide), a good metal chelate, led us to a new, efficient, mixed inhibitor for mushroom tyrosinase. Another important feature pointed out by our study is the presence of more than one site for aurone compounds on mushroom tyrosinase. Because study of the binding of the hybrid aurone was difficult to perform with the enzyme, we undertook binding studies with tyrosinase functional models in order to elucidate the binding mode (chelating vs. bridging) on a dicopper(II) center. Use of EPR combined with theoretical DFT calculations allowed us to propose a preferred chelating mode for the interaction of the hybrid aurone with a dicopper(II) center.

Inhibitor development: We have highlighted the extraordinary versatile effects of the aurone structure on mushroom Ty activity. Depending on the position of the OH group on the B-ring, aurones can behave either as substrates or as activator. We also synthesized and evaluated a compound combining an aurone moiety with HOPNO, an efficient inhibitor for mushroom Ty (K_iu=1.62 μM and K_ic=1.27 μM).

Intramolecular ligation of peptide hydrazides is reported to occur readily, causing the lactamization of fully unprotected peptides in an epimerization-free manner. This method relies on the routine procedures of Fmoc solid-phase peptide synthesis. It can be used to prepare cyclic peptides and cyclic proteins under simpler, mild conditions at lower costs.

Positively constrained: The first examples of photocontrolled RNA binding peptides are described. The large number of positively charged sides chains in the Rev response element (RRE) of an HIV type I targeting α-helix imposes constraints on the choice of azobenzene-derived crosslinker.

A major limitation of solution NMR is molecular tumbling, which is often too slow for detection. Here we demonstrate that solid-state NMR spectroscopy in combination with flash freezing of cells can be used to detect proteins in the cellular environment and provides information on backbone chemical shifts.

Isoprenoids form the largest family of compounds found in nature. Isoprenoids are often attached to other moieties such as aromatic compounds, indoles/tryptophan, and flavonoids. These reactions are catalyzed by three phylogenetically distinct prenyltransferases: soluble aromatic prenyltransferases identified mainly in actinobacteria, soluble indole prenyltransferases mostly in fungi, and membrane-bound prenyltransferases in various organisms. Fusicoccin A (FC A) is a diterpene glycoside produced by the plant-pathogenic fungus Phomopsis amygdali and has a unique O-prenylated glucose moiety. In this study, we identified for the first time, from a genome database of P. amygdali, a gene (papt) encoding a prenyltransferase that reversibly transfers dimethylallyl diphosphate (DMAPP) to the 6′-hydroxy group of the glucose moiety of FC A to yield an O-prenylated sugar. An in vitro assay with a recombinant enzyme was also developed. Detailed analyses with recombinant PAPT showed that the enzyme is likely to be a monomer and requires no divalent cations. The optimum pH and temperature were 8.0 and 50 °C, respectively. K_m values were calculated as 0.49±0.037 μM for FC P (a plausible intermediate of FC A biosynthesis) and 8.3±0.63 μM for DMAPP, with a k_cat of 55.3±3.3×10⁻³ s. The enzyme did not act on representative substrates of the above-mentioned three types of prenyltransferase, but showed a weak transfer activity of geranyl diphosphate to FC P.

A novel sugar prenyltansferase, PAPT, from the fungus Phomopsis amygdali has been cloned and characterized. This enzyme transfers dimethylallyl diphosphate to the 6′-hydroxy group of the glucose moiety of fusicoccin (FC) A, a diterpene glucoside. To the best of our knowledge, this is the first enzyme to catalyze prenylation of a hydroxyl group in a glucose moiety.

Route of the sun block: According to empirical evidence, sun-screening mycosporine-like amino acids (MAAs) in Eukarya originate from the shikimic acid pathway, whereas in cyanobacteria, biosynthesis of the MAA shinorine reportedly occurs through the pentose phosphate pathway. However, gene deletion shows that the cyanobacterium Anabaena variabilis ATCC 29143 does not biosynthesise shinorine exclusively by this route.

A FlAsH of potential: The specific binding of the biarsenical probe to the tetracysteine motif has matured as a tool for cell biology studies. Combining two such binders in one probe generates a useful reporter of protein dimerization events. The current state of art and the perspective for future developments are highlighted.

Lasso peptides: unique structure and unusual stability: The highly ordered structures of lasso peptides comprising 16–21 amino acids render them unusually stable. The extremely neat 2D NMR spectra obtained in organic solvents makes NMR a powerful tool for determining these lasso structures.

Inversion of stereoselectivity: screening of a minimal mutant library revealed a cytochrome P450 BM3 variant M01 A82W S72I capable of producing 16 α-OH-testosterone. Remarkably, a single active site mutation S72I in M01 A82W inverted the stereoselectivity of hydroxylation from 16 β to 16 α. Introduction of S72I mutation in another 16 β-OH-selective variant M11 V87I, also resulted in similar inversion of stereoselectivity.

Protein–protein interactions (PPIs) are central to biological processes and represent an important class of therapeutic targets. Here we show that the interaction between FK506-binding protein 12 fused to green fluorescent protein (GFP-FKBP) and the rapamycin-binding domain of mTor fused to Escherichia coli dihydrofolate reductase (FRB-eDHFR) can be sensitively detected (signal-to-background ratio (S/B)>100) and accurately quantified within an impure cell lysate matrix using a luminescence resonance energy transfer (LRET) assay. Ascomycin-mediated inhibition of GFP-FKBP–rapamycin–FRB-eDHFR complex formation was also detected at high S/B ratio (>80) and Z′-factor (0.89). The method leverages the selective, stable binding of trimethoprim (TMP)-terbium complex conjugates to eDHFR, and time-resolved, background-free detection of the long-lifetime (~ms) terbium-to-GFP LRET signal that indicates target binding. TMP–eDHFR labeling can be adapted to develop high-throughput screening assays and complementary, quantitative counter-screens for a wide variety of PPI targets with a broad range of affinities that may not be amenable to purification.

Assay of a lifetime: Noncovalent binding of a trimethoprim-linked Tb³⁺ complex (TMP-Tb) to E. coli dihydrofolate reductase (eDHFR) enables a highly sensitive, luminescent resonance energy transfer (LRET) assay to detect and measure protein–protein interactions and their inhibition.

New HyTs are a knockout: We previously reported that labeling HaloTag proteins with low molecular weight hydrophobic tags (HyTs) leads to targeted degradation of HaloTag fusion proteins. In this report, we employed a chemical approach to extend this hydrophobic tagging methodology to highly stabilized proteins by synthesizing and evaluating a library of HyTs, which led to the identification of HyT36.

Spot lit: Photocaged nucleic acids have been used to regulate gene expression through the action of light. Whereas most methods target mRNAs, DNA decoys have recently been used to target DNA transcription by targeting specific DNA-transcription-factor interactions. This has allowed researchers to “turn-off” transcription through the action of light on caged nucleic acids for the first time.

The role of glycosylation of proteins on its binding affinity is not well understood. Even a monosaccharide (magenta) placed at a glycosylation site can significantly enhance binding of peptides to their receptor. If glycosylated, an HIV protein binds stronger and faster to its primary receptors on human cells.

“No man is an island.”^[1] With apologies to John Donne, the same could be said for a bacterium. The discovery of bacterial quorum sensing and its relevance to microbial ecology and pathogenesis have fueled the increasing scrutiny of the molecular mechanisms responsible for the apparent group behavior of microbes.^[2] A number of chemically diverse small molecules act as diffusible signaling molecules that regulate gene expression in a population-dependent manner. Some of these signals, such as the N-acyl-L-homoserine lactones, are produced and sensed by others in the same or closely related species, and other chemical classes of signals are used more broadly for interspecies and even interkingdom communication.^[3] As a field, the study of these microbial social networks has been termed “sociomicrobiology.”^[4]

Send to know: A recent publication by Garner, Janda and co-workers develops a novel, extensible, fluorescent, dendrimeric probe for detecting AI-2 receptors, now enabling the imaging of quorum-sensing receptors in a wide variety of bacteria without the use of reporter genes.

Wiley, Hoboken 2011, XIV+351 pp., hardcover $ 110.00.—ISBN 978-0-470-72193-3

The cover picture shows the human blood group B galactosyltransferase that resides in the Golgi apparatus. Design and synthesis of inhibitors of glycosyltransferases is still challenging. Here, the binding site of the enzyme is occupied by a pentityl conjugate of uric acid. Structure-based design and synthesis led to nonionic UDP mimics that act as enzyme inhibitors. For more information, see the Full Paper by B. Meyer et al. on p. 443 ff. The illustration of the Golgi apparatus is taken from U. Lüttge, M. Kluge, G. Thiel, Botanik–Die umfassende Biologie der Pflanzen, Wiley-VCH, Weinheim, 2010.

The serine protease plasmin is ubiquitously expressed throughout the human body in the form of the zymogen plasminogen. Conversion to active plasmin occurs through enzymatic cleavage by plasminogen activators. The plasminogen activator/plasmin system has a well-established function in the removal of intravascular fibrin deposition through fibrinolysis and the inhibition of plasmin activity; this has found widespread clinical use in reducing perioperative bleeding. Increasing evidence also suggests diverse, although currently less defined, roles for plasmin in a number of physiological and pathological processes relating to extracellular matrix degradation, cell migration and tissue remodelling. In particular, dysregulation of plasmin has been linked to cancer invasion/metastasis and various chronic inflammatory conditions; this has prompted efforts to develop inhibitors of this protease. Although a number of plasmin inhibitors exist, they commonly suffer from poor potency and/or specificity of inhibition that either results in reduced efficacy or prevents clinical use. Consequently, there is a need for further development of high-affinity plasmin inhibitors that maintain selectivity over other serine proteases. This review summarises clearly defined and potential applications for plasmin inhibition. The properties of naturally occurring and engineered plasmin inhibitors are discussed in the context of current knowledge regarding plasmin structure, specificity and function. This includes design strategies to obtain the potency and specificity of inhibition in addition to controlled temporal and spatial distribution tailored for the intended use.

The serine protease plasmin plays a pivotal role in a variety of processes including fibrinolysis, degradation of the extracellular matrix and cell migration. This review focuses on naturally occurring and engineered inhibitors of this important enzyme.

(R)evolution of protein-based calcium sensors: Expanding the toolbox of genetically encoded calcium sensors with new colors and traits is important for understanding calcium signaling and its relation to other intracellular pathways. Campbell and co-workers have used a new directed-evolution strategy to develop a rich palette of new sensors, including the first red-shifted, genetically encoded calcium sensor.

The sialome comprises sialylated glycoproteins and glycolipids that play essential roles in cell–cell communication. Using azide-modified molecular precursors of sialic acids and copper-free click chemistry, we visualized the spatiotemporal dynamics of the sialome in live zebrafish embryos.

Needles and noodles: Studying amyloid toxicity is important for understanding protein misfolding diseases. Using a luminescent conjugated polythiophene, we found that cell binding of nontoxic filamentous amyloids of insulin and β2-microglobulin was less efficient than that of toxic fibrillar amyloids; this suggests a correlation between amyloid toxicity and cell binding.

Sticky residue: Pyrroline-carboxy-lysine (Pcl) can be readily incorporated into proteins expressed in E. coli and mammalian cells by using the pyrrolysyl tRNA/tRNA synthetase pair. Pcl can be used as a single amino acid purification tag and can be site-specifically modified with functional probes during the elution process (see scheme).

Roped in: The lasso peptide microcin J25 (MccJ25) is matured by two enzymes and is exported by a putative ABC transporter (see graphic). We probed the function of the maturation enzymes using mutagenesis. We demonstrate that fusions of the enzymes with intervening linkers can produce MccJ25. Even a 151 kDa tripartite fusion between the ABC transporter and the two enzymes is capable of producing and exporting MccJ25.

Microcin J25 is a potent antibacterial peptide produced by Escherichia coli AY25. It displays a lasso structure, which consists of a knot involving an N-terminal macrolactam ring through which the C-terminal tail is threaded and sterically trapped. In this study, we rationally designed and performed site-specific mutations in order to pinpoint the sequence determinants of the lasso topology. Structures of the resulting variants were analysed by a combination of methods (mass spectrometry, NMR spectroscopy, enzymatic digestion), and correlated to the antibacterial activity. The selected mutations resulted in the production of branched-cyclic or lasso variants. The C-terminal residues below the ring (Tyr20, Gly21) and the size of the macrolactam ring were revealed to be critical for both the lasso scaffold and bioactivity, while shortening the loop region (Tyr9–Ser18) or extending the C-terminal tail below the ring did not alter the lasso structure, but differentially affected the antibacterial activity. These results provide new insights for the bioengineering of antibacterial agents using a lasso peptide as template.

Rope tricks: Our comprehensive structure–activity analysis of the antimicrobial lasso peptide microcin J25 shows which sequence elements govern 1) the stabilization of the lasso fold and 2) the antibacterial activity of the peptide.

DNAzymes are catalytically active DNA molecules that use metal cofactors for their enzymatic functions. While a growing number of DNAzymes with diverse functions and metal selectivities have been reported, the relationships between metal ion selectivity, conserved sequences and structures responsible for selectivity remain to be elucidated. To address this issue, we report biochemical assays of a family of previously reported in vitro selected DNAzymes. This family includes the clone 11 DNAzyme, which was isolated by positive and negative selection, and the clone 18 DNAzyme, which was isolated by positive selection alone. The clone 11 DNAzyme has a higher selectivity for Co²⁺ over Pb²⁺ compared with clone 18. The reasons for this difference are explored here through phylogenetic comparison, mutational analysis and stepwise truncation. A novel DNAzyme truncation method incorporated a nick in the middle of the DNAzyme to allow for truncation close to the nicked site while preserving peripheral sequences at both ends of the DNAzyme. The results demonstrate that peripheral sequences within the substrate binding arms, most notably the stem loop, loop II, are sufficient to restore its selectivity for Co²⁺ over Pb²⁺ to levels observed in clone 11. A comparison of these sequences' secondary structures and Co²⁺ selectivities suggested that metastable structures affect metal ion selectivity. The Co²⁺ selectivity of the clone 11 DNAzyme showed that the metal ion binding and selectivities of small, in vitro selected DNAzymes may be more complex than previously appreciated, and that clone 11 may be more similar to larger ribozymes than to other small DNAzymes in its structural complexity and behavior. These factors should be taken into account when metal-ion selectivity is required in rationally designed DNAzymes and DNAzyme-based biosensors.

Peripheral sequences change DNAzyme selectivity: A stem loop and nucleotides within peripheral sequences influence the Co²⁺ selectivity of the clone 11 DNAzyme. An alternative truncation approach provides insights into structure function relationships of metal-dependent DNAzymes.

In a cell-based assay for novel inhibitors, we have discovered that two glycosides of 5-thiomannose, each containing an interglycosidic nitrogen atom, prevented the correct zymogen processing of the prohormone proopiomelanocortinin (POMC) and the transcription factor sterol-regulatory element-binding protein-2 (SREBP-2) in mouse pituitary cells and Chinese hamster ovary (CHO) cells, respectively. In the case of SREBP-2, these effects were correlated with the altered N-linked glycosylation of subtilisin/kexin-like isozyme-1 (SKI-1), the protease responsible for SREBP-2 processing under sterol-limiting conditions. Further examination of the effects of these compounds in CHO cells showed that they cause extensive protein hypoglycosylation in a manner similar to type I congenital disorders of glycosylation (CDGs) since the remaining N-glycans in treated cells were complete (normal) structures. The under-glycosylation of glycoproteins in 5-thiomannoside-treated cells is now shown to be caused by the compromised biosynthesis of the dolichol-linked oligosaccharide (DLO) N-glycosylation donor, although the nucleotide sugars required for the synthesis of DLOs were neither reduced under these conditions, nor were their effects reversed upon the addition of exogenous mannose. Analysis of DLO intermediates by fluorophore-assisted carbohydrate electrophoresis demonstrated that 5-thiomannose-containing glycosides block DLO biosynthesis most likely at a stage prior to the GlcNAc₂Man₃ intermediate, on the cytosolic face of the endoplasmic reticulum.

It's own sweet ways: Disaccharide analogues containing nonreducing 5-thiomannose moieties (see structures) have recently been shown to affect the N-glycosylation and activity of proprotein convertases. We have discovered that these compounds inhibit the early steps of dolichol-linked oligosaccharide biosynthesis, and thereby reduce protein N-glycosylation in a manner that resembles congenital disorders of glycosylation.

Several proteases like the high temperature requirement A (HtrA) protein family containing internal or C-terminal PDZ domains play key roles in protein quality control in the cell envelope of Gram-negative bacteria. While several HtrA proteases have been extensively characterized, many features of C-terminal processing proteases such as tail-specific protease (Tsp) are still unknown. To fully understand these cellular control systems, individual domains need to be targeted by specific peptides acting as activators or inhibitors. Here, we describe the identification and design of potent inhibitors and activators of Tsp. Suitable synthetic substrates of Tsp were identified and served as a basis for the generation of boronic acid-based peptide inhibitors. In addition, a proteomic screen of E. coli cell envelope proteins using a synthetic peptide library was performed to identify peptides capable of amplifying Tsp's proteolytic activity. The implications of these findings for the regulation of PDZ proteases and for future mechanistic studies are discussed.

Turning Tsp ON: Chemical biology approaches have revealed that the quality control protease Tsp is triggered into an active state via interactions with various activating clues.

Elevated expression of interleukin-8 (IL-8) has been implicated in inflammatory diseases, in tumor growth, and in angiogenesis. The aim of this study was to identify natural or synthetic compounds that suppress IL-8 production in response to interleukin-1 (IL-1), the natural inflammatory stimulus of the IL-8 gene. We therefore developed an IL-1-inducible cell-based screening assay by stable integration of an IL-8 reporter gene into HeLa S3 cells. The screening of heterogeneous compound libraries revealed several compounds that displayed an inhibitory effect on the reporter gene expression. Following hit validation, we focused on the most efficient compound, spirangien A, and its chemical derivate spirangien M522. Detailed analysis shows that both compounds are potent inhibitors of the endogenous IL-8 gene transcription. Furthermore, both compounds decelerate the phosphorylation and degradation of IκBα, the key regulator of the IL-1-stimulated NF-κB signaling pathway. Our study has identified the two spirangiens A and M522 as potent inhibitors of IL-1/NF-κB-mediated IL-8 gene expression.

The screening of heterogeneous compound libraries identified the myxobacterial compound spirangien A and its derivate spirangien M522 as potent inhibitors of IL-8 expression. Both compounds decelerate the phosphorylation and degradation of IκBα, the key regulator of the IL-1-stimulated NF-κB signaling pathway.

The field of bacterial natural product research is currently undergoing a paradigm change concerning the discovery of natural products. Previously most efforts were based on isolation of the most abundant compound in an extract, or on tracking bioactivity. However, traditional activity-guided approaches are limited by the available test panels and frequently lead to the rediscovery of already known compounds. The constantly increasing availability of bacterial genome sequences provides the potential for the discovery of a huge number of new natural compounds by in silico identification of biosynthetic gene clusters. Examination of the information on the biosynthetic machinery can further prevent rediscovery of known compounds, and can help identify so far unknown biosynthetic pathways of known compounds. By in silico screening of the genome of the myxobacterium Stigmatella aurantiaca Sg a15, a trans-AT polyketide synthase/non-ribosomal peptide synthetase (PKS/NRPS) gene cluster was identified that could not be correlated to any secondary metabolite known to be produced by this strain. Targeted gene inactivation and analysis of extracts from the resulting mutants by high performance liquid chromatography coupled to high resolution mass spectrometry (HPLC-HRMS), in combination with the use of statistical tools resulted in the identification of a compound that was absent in the mutants extracts. By matching with our in-house database of myxobacterial secondary metabolites, this compound was identified as rhizopodin. A detailed analysis of the rhizopodin biosynthetic machinery is presented in this manuscript.

New methodologies for efficient genome mining of secondary metabolites are of the utmost importance to tap the full potential revealed by the ever-expanding sequence data. Targeted gene inactivation combined with sophisticated analytical and statistical tools has led to the identification and characterisation of the rhizopodin biosynthetic gene cluster.

Primary alcohol oxidation by aryl-alcohol oxidase (AAO), a flavoenzyme providing H₂O₂ to ligninolytic peroxidases, is produced by concerted proton and hydride transfers, as shown by substrate and solvent kinetic isotope effects (KIEs). Interestingly, when the reaction was investigated with synthesized (R)- and (S)-α-deuterated p-methoxybenzyl alcohol, a primary KIE (≈6) was observed only for the R enantiomer, revealing that the hydride transfer is highly stereoselective. Docking of p-methoxybenzyl alcohol at the buried crystal active site, together with QM/MM calculations, showed that this stereoselectivity is due to the position of the hydride- and proton-receiving atoms (flavin N5 and His502 Nε, respectively) relative to the alcohol Cα-substituents, and to the concerted nature of transfer (the pro-S orientation corresponding to a 6 kcal mol⁻¹ penalty with respect to the pro-R orientation). The role of His502 is supported by the lower activity (by three orders of magnitude) of the H502A variant. The above stereoselectivity was also observed, although activities were much lower, in AAO reactions with secondary aryl alcohols (over 98 % excess of the R enantiomer after treatment of racemic 1-(p-methoxyphenyl)ethanol, as shown by chiral HPLC) and especially with use of the F501A variant. This variant has an enlarged active site that allow better accommodation of the α-substituents, resulting in higher stereoselectivity (S/R ratios) than is seen with AAO. High enantioselectivity in a member of the GMC oxidoreductase superfamily is reported for the first time, and shows the potential for engineering of AAO for deracemization purposes.

Stereoselectivity and concerted transfers: Enantioselective hydride abstraction from alcohol substrates in a member of the GMC superfamily is reported. We suggest that this is due to oxidation by aryl-alcohol oxidase; in contrast to other GMCs, this involves hydride abstraction by flavin N5 with concerted proton abstraction by catalytic His502, as shown by KIEs and QM/MM calculations.

The binding of nucleosides to abasic site (AP site)-containing DNA duplexes (AP-DNAs) carrying complementary nucleosides opposite the AP site was investigated by thermal denaturation and isothermal titration calorimetric (ITC) experiments. Purine nucleosides show high affinities (K_d=14.1 μM for adenosine and 41.8 μM for guanosine) for binding to the AP-DNAs, and the interactions are driven primarily by the enthalpy change, similarly to the case of DNA intercalators. In contrast, pyrimidine nucleosides do not show noticeable binding to the AP-DNAs, thus suggesting that stacking interaction at the AP site plays a key role in the binding of purine nucleosides to the AP-DNAs, as revealed by ITC measurements. Next, to apply an AP-DNA as an aptasensor for adenosine, a competitive assay between adenosine and AP-site-binding fluorescent ligand was performed. The assay employs a fluorescent ligand, riboflavin, that binds to the AP site in a DNA duplex, thereby causing fluorescence quenching. By adding adenosine to the riboflavin/AP-DNA complex, the binding of adenosine to the AP site causes release of riboflavin from the AP site, thereby resulting in restoration of riboflavin fluorescence. AP-DNAs can serve as a new class of aptasensors—a limit of detection of 0.7 μM was obtained for adenosine. In contrast to conventional aptasensors for adenosine, the present method shows high selectivity for adenosine over the other nucleotides (AMP, ADP and ATP). The method does not require covalent labelling of fluorophores, and thus it is cost-effective; finally, the method was successfully demonstrated to be applicable for the detection of adenosine in horse serum.

Purine nucleosides bind to the AP site in DNA duplexes carrying complementary nucleosides opposite the AP site, whereas pyrimidine nucleosides do not show noticeable binding. By using this characteristic binding feature, aptasensing of adenosine is attained by competitive binding between adenosine and riboflavin bound to the AP site in a DNA duplex.

Glycosyltransferases play an important role in the formation of oligosaccharides and glycoconjugates. To find suitable and selective inhibitors for this class of enzymes is still challenging. Here, we describe a novel concept that allows the design of inhibitors based on the structure of the donor substrate binding pocket. As a first step we describe the design, synthesis and analysis of inhibitors of the human blood group B galactosyltransferase (GTB). This enzyme served as a model system to study the concept, which can be used for easy access of glycosyltransferase inhibitors in general. In silico docking of bicyclic heteroaromatic ligands to GTB and experimental verification of binding affinities by saturation transfer difference NMR (STD NMR) spectroscopy gave 9-N-pentityl uric acid derivatives as non-ionic mimics of UDP. Two derivatives were synthesized and showed inhibitory activity for GTB as determined by competitive STD NMR experiments and by a radiolabeled enzyme assay.

Inhibitors of glycosyltransferases are important tools for studying sugar transfer to a variety of natural product based acceptors. We present a new concept for specific inhibitors of glycosyltransferases. As a first step we present an inhibitor for the UDP binding site of the human blood group B galactosyltransferase (see graphic).

Gene knock-out of C-type lectin receptors expressed in dendritic cells induced significant alteration of serum N-glycans compared with that of gender-matched controls. Glycotyping analysis suggested that putative-core fucosylation is strongly influenced by differences in the dominant mechanisms after carbohydrate recognition by pattern-recognition receptors, endocytosis of ligands, or induction of cytokines/chemokines. However, the loss of galectin-9, a ligand for T-helper type 1-specific cell-surface molecule, did not affect most N-glycan profiles. Interestingly, lack of the Chst3 gene (chondroitin 6-sulfotransferase) appeared to influence markedly the expression of most N-glycans, especially highly modified glycoforms bearing multiple Neu5Gc, Fuc, and LacNAc units. In contrast, genetic mutations in B4galnt1 and B4galnt2 (GalNAc transferase, responsible for the synthesis of many gangliosides) induced no discernable alteration. These results indicate that the biosynthesis of N-glycans of serum glycoproteins can be affected not only by direct genetic mutations in the glycosyltransferases but also by changes in metabolite availability in sugar nucleotide synthesis and Golgi N-glycosylation pathways caused concertedly in whole cells, tissues, and organs by milder deficiencies in immune cell-surface lectins. Many common chronic conditions, such as autoimmunity, metabolic syndrome, and aging/dementia result.

Glycotyping of KO mice: Glycotyping analysis of serum N-glycomes from gene-knockout mice that show no significant phenotype or abnormality can provide highly informative data to facilitate further comprehensive discussion of functions of cell surface C-type lectins in the maintenance of homeostatic balance of general immune system.

Twenty-two β-resorcylic acid lactones (RALs) were evaluated for cytotoxicity against human breast cancer cells to find their structure–activity relationship (SAR). Monocillin II, a trans-enone RAL without epoxy and conjugated dienone, was found to have higher activity in inhibiting tumor cell growth in both in vitro experiment and in vivo nude xenografted mice model than its analogue radicicol, an anticancer lead compound. We demonstrated for the first time that monocillin II could arrest breast cancer cell cycle in G1 phase, which might partially be the result of its inhibition effect on the phosphorylation of the Thr160 residue of cyclin dependent kinase 2 (CDK2), a key enzyme in cell-cycle regulation. Moreover, monocillin II exhibited inhibition of heat shock protein 90 (Hsp90) and depleted its target proteins, Raf-1 and A-Raf, which are involved in Ras/Raf/MEK/ERK mitogen-activated protein kinase (MAPK) pathway. Remarkably, we found that monocillin II could inhibit activation of MAPKs including ERK, JNK and p38, which might be involved in the inactivation of CDK2. These results suggest that monocillin II has potential therapeutic benefits in breast cancer prevention and intervention.

From primary screening for anti-breast-cancer agents, a neglected RAL, monocllin II, was found to have extraordinary activity both in vitro and in vivo. Its underlying mechanisms of action were investigated, and some interesting results were discovered. The high activity of monocillin II was assumed to derive from its chemical structure when compared with its anologue, radicicol.

Mitochondrially targeted agents have the capacity to be both vehicles for the delivery of bioactive agents and mitochondrial disrupters and show promise for the treatment of various diseases. Engineering these agents to specifically accumulate or disrupt the mitochondrion is challenging, as there is a fine line between characteristics of the molecules that accomplish each task. Here, we assess the physicochemical properties governing mitochondrial matrix accumulation or membrane disruption caused by mitochondria-penetrating peptides. Increases in peptide length and hydrophobicity were uncovered as the dominant factors in deriving membrane disruptive activity. Shorter, less hydrophobic peptides did not disrupt the mitochondrial membrane, but rather accumulated in the mitochondrial matrix without interfering with cellular activity. These shorter peptides, however, can trigger cytochrome c release through activation of the permeability transition pore complex (PTPC), but only at very high concentrations. This study illustrates that the activity of a mitochondria-localizing agent can be controlled through alterations in peptide hydrophobicity and dosing concentrations.

Decommissioning the powerhouse: Mitochondrial-targeting agents have the capacity to be both vehicles for the delivery of bioactive agents and mitochondrial disrupters and show promise for the treatment of various diseases. Here, we assess the physicochemical properties governing mitochondrial matrix accumulation or membrane disruption caused by mitochondria-penetrating peptides (see graphic).