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ChemBioChem - published by Wiley
ChemBioChem is a source for important primary and secondary information across the whole field of chemical biology, bio(in)organic chemistry, and biochemistry. Ist mission is to integrate this wide and flourishing field, ranging from complex carbohydrates through peptides/proteins to DNA/RNA, from combinatorial chemistry and biology to signal transduction, from catalytic antibodies to protein folding, from bioinformatics and structural biology to drug design
Thymine glycol (Tg), one of the oxidized bases formed in DNA by reactive oxygen species, is repaired by the DNA glycosylases such as NEIL1, NTH1 and Endo III. In our recent studies, we showed that NEIL1's catalytic efficiency and lesion specificity are regulated by an RNA-editing adenosine deamination reaction. In this study, we synthesized oligodeoxynucleotides containing 2?-fluorothymidine glycol with either ribo or arabino configuration and investigated the binding of these modified DNAs with the unedited and edited forms of human NEIL1 along with E. coli Endo III. For the two forms of hNEIL1, binding affinities to FTg-containing DNA were similar indicating that the editing effect is more subtle than to simply alter substrate affinity. While the NEIL1-binding to FTg-containing DNAs was largely insensitive to C5 and 2? stereochemistry, a preference was observed for the FTg–G pair over the FTg–A pair. In addition, we found that optimal binding is observed with Endo III and duplex DNA with riboFTg(5S) paired with dG. The modified DNAs reported here will provide useful tools for further characterizing the interaction between DNA repair glycosylases and thymine glycol containing DNA.DNA repair and RNA editing: Oligodeoxynucleotides containing 2?-fluorothymidine glycol with either ribo or arabino configuration were synthesized and their binding with the unedited and edited forms of hNEIL1 along with Endo III were investigated. For both forms of hNEIL1, the binding affinities were similar, unlike the significant difference in glycosylase activity between the two forms of NEIL1.
The immunomodulatory glycolipid ?-galactosylceramide (?-GalCer) binds to CD1d and exhibits potent activity as a ligand for invariant CD1d-restricted natural killer-like T cells (iNKT cells). Structural analogues of ?-GalCer have been synthesised to determine which components are required for CD1d presentation and iNKT cell activation, however, to date the importance of the phytosphingosine 4-hydroxyl for iNKT cell activation has been disputed. To clarify this, we synthesised two 4-deoxy ?-GalCer analogues (sphinganine and sphingosine) and investigated their ability to activate murine and human iNKT cells. Analysis revealed that the analogues possessed comparable activity to ?-GalCer in stimulating murine iNKT cells, but were severely compromised in their ability to stimulate human iNKT cells. Here we determined that species-specific glycolipid activity was due to a lack of recognition of the analogues by the T-cell receptors on human iNKT cells rather than insufficient presentation of the analogues on human CD1d molecules. From these results we suggest that glycolipids developed for potent iNKT cell activity in humans should contain a phytosphingosine base.Two 4-deoxy analogues of ?-GalCer were synthesised and their species-specific activities investigated. Here, we show that the 4-hydroxyl of ?-GalCer is critical for the activation of human, but not murine, iNKT cells. This species-specific activity is due to a lack of human iNKT cell recognition, rather than insufficient CD1d presentation.
Caulobacter crescentus CcrM is a DNA-(adenine N6)-methyltransferase that methylates adenine in the sequence GANTC with high specificity. To investigate its mechanism of DNA recognition, we used the crystal structure of a related methyltransferase (M1.MboII, which modifies GAAGA) as a starting point, and docked into it a DNA substrate to identify the protein regions that approach the DNA. After alignment of CcrM and M1.MboII, we identified four candidate regions in CcrM to contain residues involved in DNA recognition. We mutated 20 amino acid residues within these regions, purified the CcrM variants, and determined their DNA-binding and catalytic activity on a cognate GANTC substrate and on nine near-cognate substrates, each of which contained a single base-pair substitution in the recognition sequence. Altogether, we identified four residues in two of the regions, mutations of which resulted in a strong (>100-fold) reduction of methylation activity. Our data show that DNA recognition by CcrM is a cooperative process, because disruption of critical contacts led to loss of catalytic activity but not to a relaxation in specificity. In addition, we identified a change in the readout of the fifth base pair in the GANTC sequence with two other CcrM variants that showed smaller reductions in overall activity. Based on this and the sequence alignment of CcrM with other DNA methyltransferases of same or related recognition sequence, we propose roles for these two regions in DNA recognition by CcrM.DNA recognition by the CcrM DNA methyltransferase was studied by protein modeling and site-directed mutagenesis of 20 amino acid residues. Four residues in two loops of the protein were identified to be important for DNA recognition by CcrM, which occurs in a very cooperative process.
A series of 18-mer peptide nucleic acids (PNAs) targeted against micro-RNA miR-210 was synthesised and tested in a cellular system. Unmodified PNAs, R8-conjugated PNAs and modified PNAs containing eight arginine residues on the backbone, either as C2-modified (R) or C5-modified (S) monomers, all with the same sequence, were compared. Two different models were used for the modified PNAs: one with alternated chiral and achiral monomers and one with a stretch of chiral monomers at the N terminus. The melting temperatures of these derivatives were found to be extremely high and 5?M urea was used to assess differences between the different structures. FACS analysis and qRT-PCR on K562 chronic myelogenous leukaemic cells indicated that arginine-conjugated and backbone-modified PNAs display good cellular uptake, with best performances for the C2-modified series. Resistance to enzymatic degradation was found to be higher for the backbone-modified PNAs, thus enhancing the advantage of using these derivatives rather than conjugated PNAs in the cells in serum, and this effect is magnified in the presence of peptidases such as trypsin. Inhibition of miR-210 activity led to changes in the erythroid differentiation pathway, which were more evident in mithramycin-treated cells. Interestingly, the anti-miR activities differed with use of different PNAs, thus suggesting a role of the substituents not only in the cellular uptake, but also in the mechanism of miR recognition and inactivation. This is the first report relating to the use of backbone-modified PNAs as anti-miR agents. The results clearly indicate that backbone-modified PNAs are good candidates for the development of very efficient drugs based on anti-miR activity, due to their enhanced bioavailabilities, and that overall anti-miR performance is a combination of cellular uptake and RNA binding.Chiral PNAs containing backbone arginine residues have been shown to display good anti-miR activities against miR-210, thus affecting cellular differentiation because they display strong RNA-binding properties, good cellular permeation and resistance to peptidases. We show that the stereochemistry, type of modification and distribution of modified monomers affect the overall anti-miR properties.
Smooth converter: Bioconjugation of superfolder GFP involving the formation of an unusually stable, and unprecedented, cyclic sulfonium species is described. This sulfonium can undergo smooth reaction with a range of nucleophiles to give sulfur-, selenium- and azide-modified GFP derivatives in high conversions.
The human immunodeficiency virus type-1 (HIV-1) is able to shield immunogenic peptide epitopes on its envelope spike (a trimer of two glycoproteins, gp120 and gp41) by presenting numerous host-derived N-linked glycans. Nevertheless, broadly neutralizing antibodies against gp120 and gp41 have been isolated from HIV-1-infected patients and provide protection against viral challenge in animal models. Among these, the monoclonal antibody 2G12 binds to clusters of high-mannose-type glycans that are present on the surface of gp120. These types of glycans have thus been envisaged as target structures for the development of synthetic agents capable of eliciting 2G12-like antibodies. High-resolution structural studies of 2G12 and chemically defined glycan-type ligands, including crystallographic data, have been performed to gain an insight into this interaction. Further studies are still required to design a carbohydrate-based vaccine for HIV. Our previous NMR studies highlighted different recognition modes of two branched synthetic oligosaccharides, a penta- and a heptamannoside, by 2G12 in solution. In order to clarify the underlying structural reasons for such different behaviors, we have herein “dissected” the branches into the linear tri- and tetra- oligomannosides by chemical synthesis and studied their interactions with 2G12 in solution by saturation transfer difference (STD) NMR spectroscopy. The results confirm the distinct preferences of 2G12 for the studied branches and afford explanations for the observed differences. This study provides important structural information for further ligand optimizations. Possible effects of structural modifications on the solvent-exposed end of the ligands are also discussed.Sweet gp120 arms: Molecular recognition of synthetic linear tri- and tetramannosides by the anti-HIV-1 antibody 2G12 was studied by NMR spectroscopy. These sugar ligands mimic the arms of two higher branched oligomannosides that contain structural motifs from natural gp120 glycans. Their binding epitopes and affinities for 2G12 were determined and compared to previous studies.
Growing interest in the structure of G-quartets is hampered by the problem of polymorphism. Use of a template to secure the guanines into an organized array can circumvent this problem. We report the first crystal structure of a template-assembled G-quartet. The structure is comparable to those of natural G-quartets, but it also shows quartet–quartet interactions where there is no covalent linkage between the quartets.
The genetic incorporation of one azide-containing and one keto-containing noncanonical amino acid into a protein at amber and ochre mutation sites respectively, followed by their orthogonal reactions with hydroxylamine-containing and cyclooctyne-containing dyes allows highly efficient one-pot site-specific dual labeling of the protein in a catalyst-free fashion.
The inside cover picture shows an aptasensor based on “DNA traffic lights”: On p. 1136?ff., H.-A. Wagenknecht and C. Holzhauser explain how the green emitter Thiazole Orange and the red emitter Thiazole Red were embedded as base surrogates in the DNA architecture. As a result, a significant green-to-red fluorescence change occurred upon specific target binding.
The cover picture shows the structure of the class III lantibiotic LabA2, which displays remarkable antiviral as well as analgesic activities in a neuropathic mouse model. A special feature in its structure is the unusual ?,?-disubstituted amino acid labionin. Interestingly LabA2-homologous gene clusters can also be found in a great number of other Actinomycetes such as the model Actinomycete Streptomyces coelicolor and the corresponding ram gene cluster. A MS-based screening led to the discovery of three new representatives of class III lantibiotics and indicated stepwise leader processing by putative aminopeptidase-like protease(s) that yielded mixtures of differently processed lantibiotic peptides. Characterization by HPLC-ESI-MSMS and GC-MS suggests that these lantibiotics are a mixture of peptides, containing both lanthionine and labionin as post-translational modifications. For more information, see the paper by R. D. Süssmuth et al. on p. 1174?ff. The cover picture was prepared by Bichan Maschajechi.
Cytochrome f (Cyt f) and plastocyanin (Pc) form a highly transient complex as part of the photosynthetic redox chain. The complex from Nostoc sp. PCC 7119 was studied by NMR relaxation spectroscopy with the aim of determining the orientation of Pc relative to Cyt f. Chemical-shift-perturbation analysis showed that the presence of spin labels on the surface of Cyt f does not significantly affect the binding of Pc. The paramagnetic relaxation enhancement results are not consistent with a single orientation of Pc, thus indicating that multiple orientations must occur and suggesting that an encounter state represents a large fraction of the complex.Brief encounter: Cytochrome f and plastocyanin are redox partners in photosynthesis. Paramagnetic NMR spectroscopy was applied to characterize this complex from the cyanobacterium Nostoc sp. PCC 7119. The results show that the complex cannot be described by a single well-defined state, thus indicating that the interaction is highly dynamic.
Spontaneous, but stable: We used the intein system to generate a stable-isotope-labeled peptide. The liberated amino-terminal peptide Gln residue spontaneously converts into pyroglutamate. It might be possible to exploit this conversion to generate and subsequently characterize physiological or disease-relevant pGlu-containing peptides such as truncated A? species.
Organometallic compounds are renowned for their remarkable applications in the field of catalysis, but much less is known about their potential in chemical biology. Indeed, such compounds have long been considered to be either unstable under physiological conditions or cytotoxic. As a consequence, little attention has been paid to their possible utilisation for biological purposes. Because of their outstanding physicochemical properties, which include chemical stability, structural diversity and unique photo- and electrochemical properties, however, organometallic compounds have the ability to play a leading role in the field of chemical biology. Indeed, remarkable examples of the use of such compounds—notably as enzyme inhibitors and as luminescent agents—have recently been reported. Here we summarise recent advances in the use of organometallic compounds for chemical biology purposes, an area that we define as “organometallic chemical biology”. We also demonstrate that these recent discoveries are only a beginning and that many other organometallic complexes are likely to be found useful in this field of research in the near future.Flexible metals: This review summarises recent advances in the use of organometallic compounds in the field of chemical biology, a research area that we are defining as “organometallic chemical biology”.
Silencing prion: Copper-catalyzed transformations of prion protein (PrP) lead to the production of reactive oxygen species (ROS), PrP oxidation, and cleavage and aggregation in transmissible spongiphorm encephalopathies. Zn7MT-3 efficiently targets CuII bound in different coordination modes to PrP–CuII. By an unusual redox-dependent metal-swap reaction, MT-3 modulates the catalytic redox properties of PrP–CuII.
Making the most of your PA: Photoacoustic (PA) imaging combines the benefits of optical and ultrasound imaging. Filonov et al. have recently demonstrated the application of the genetically encoded phytochrome iRFP as a basis for cancer imaging with PA microscopy. This novel approach for imaging cancer cells opens the door to unprecedented insight into cancer progression in vivo.
Double play: The enzyme 4-oxalocrotonate tautomerase (4-OT) catalyzes not only the initial cross-coupling of acetaldehyde and benzaldehyde to yield 3-hydroxy-3-phenylpropanal, but also the subsequent dehydration of this aldol compound to yield cinnamaldehyde as the final product. Mechanism-inspired engineering provided an active site mutant (F50A) with strongly enhanced aldol condensation activity.
An unconventional dehalogenase: An engineered variant (I64V/V106L) of the mouse cytokine macrophage migration inhibitory factor (MIF) promiscuously catalyzes the hydrolytic dehalogenation of the xenobiotic organohalogen trans-3-chloroacrylic acid to acetaldehyde. Although the dehalogenase activity of this MIF variant is quite low, it achieves an ~109-fold rate enhancement, matching those of conventional enzymes acting on their natural substrates.
In order to develop an optimal subunit as a T-recognition element in hairpin polyamides, 15 novel chirality-modified polyamides containing (R)-?,?-diaminopropionic acid (R???-NH?2), (S)-?,?-diaminopropionic acid (S???-NH?2), (1R,3S)-3-aminocyclopentanecarboxylic acid (RSCp), (1S,3R)-3-amino-cyclopentanecarboxylic acid (RSCp), (1R,3R)-3-aminocyclopentanecarboxylic acid (RRCp) and (1S,3S)-3-amino-cyclopentanecarboxylic acid (SSCp) residues were synthesized. Their binding characteristics to DNA sequences 5?-TGCNCAT-3?/3?-ACGN?GTA-5? (N?N?=A?T, T?A, G?C and C?G) were systemically studied by surface plasmon resonance (SPR) and molecular simulation (MSim) techniques. SPR showed that polyamide 4, AcIm-S???-NH?2-ImPy-?-ImPy-?-Py-?Dp (?/S???-NH?2 pair), bound to a DNA sequence containing a core binding site of 5?-TGCACAT-3? with a dissociation equilibrium constant (KD) of 4.5×10?8?m. This was a tenfold improvement in specificity over 5?-TGCTCAT-3? (KD=4.5×10?7?M). MSim studies supported the SPR results. More importantly, for the first time, we found that chiral 3-aminocyclopentanecarboxylic acids in polyamides can be employed as base readers with only a small decrease in binding affinity to DNA. In particular, SPR showed that polyamide 9 (RRCp/? pair) had a 15-fold binding preference for 5?-TGCTCAT-3? over 5?-TGCACAT-3?. A large difference in standard free energy change for A?T over T?A was determined (??Go=5.9 kJ?mol?1), as was a twofold decrease in interaction energy by MSim. Moreover, a 1:1 stoichiometry (9 to 5?-TGCTCAT-3?/3?-ACGAGTA-5?) was shown by MSim to be optimal for the chiral five-membered cycle to fit the minor groove. Collectively, the study suggests that the (S)-?-amino-?-aminopropionic acid and (1R,3R)-3-aminocyclopentanecarboxylic acid can serve as a T-recognition element, and the stereochemistry and the nature of these subunits significantly influence binding properties in these recognition events. Subunit (1R,3R)-3-aminocyclopentanecarboxylic acid broadens our scope to design novel polyamides.Groovy recognition: A chiral nonaromatic five-membered cyclic subunit, (1R,3R)-3-aminocyclopentane carboxylic acid, serves as a DNA thymine-specific recognition element when incorporated into polyamides, in minor-groove sequence-specific recognition events. The results broaden our understanding and will help in the design of novel polyamides that include T-recognition preference.
We have identified the native dimer interface of heptaprenylglyceryl phosphate synthase PcrB from the bacterium Bacillus subtilis and analyzed the significance of oligomer formation for stability and catalytic activity. Computational methods predicted two different surface regions of the PcrB protomer that could be responsible for dimer formation. These bona fide interfaces were assessed both in silico and experimentally by the introduction of amino acid substitutions that led to monomerization, and by incorporation of an unnatural amino acid to allow cross-linking of the two protomers. The results showed that, in contrast to previous assumptions, PcrB uses the same interface for dimerization as the homologous geranylgeranylglyceryl phosphate synthase from Archaea. Thermal unfolding demonstrated that the monomeric proteins are only slightly less stable than wild-type PcrB. However, activity assays showed that monomerization limits the length of accepted polyprenyl pyrophosphates to three isoprene units, whereas the native PcrB substrate contains seven isoprene entities. We provide a plausible hypothesis as to how dimerization determines substrate specificity of PcrB.Working together: Many enzymes are active only when assembled as multimeric complexes, whereas a few are active both as monomers and multimers—but with different substrate specificity. We identified the dimer interface of a bacterial heptaprenylglyceryl phosphate synthase and showed that oligomer formation is crucial for binding the long-chain substrate.
Enzymatic peptide synthesis has the potential to be a viable alternative for chemical peptide synthesis. Because of the increasing commercial interest in peptides, new and improved enzymatic synthesis methods are desirable. In recently developed enzymatic strategies such as substrate mimetic approaches and enzyme-specific activation, use of the guanidinophenyl ester (OGp) group has been shown to suffer from some drawbacks. OGp esters are sensitive to spontaneous chemical hydrolysis and the group is expensive to synthesize and therefore not suitable for large-scale applications. On the basis of earlier computational studies, we hypothesized that OGp might be replaceable by simpler ester groups to make the enzyme-specific activation approach to peptide bond formation more accessible. To this end, a set of potential activating esters (Z-Gly-Act) was designed, synthesized, and evaluated. Both the benzyl (OBn) and the dimethylaminophenyl (ODmap) esters gave promising results. For these esters, the scope of a model dipeptide synthesis reaction under aqueous conditions was investigated by varying the amino acid donor. The results were compared with those obtained from a previous study of Z-XAA-OGp esters. Computational docking analysis of the set of esters was performed in order to provide insight into the differences in the reactivities of all the potential activating esters. Finally, selected ODmap- and OBn-activated amino acids were applied in the synthesis of two biologically active dipeptides on preparative scales.A set of new activating esters was evaluated in an enzymatic, papain-catalyzed approach to synthesizing dipeptides under aqueous conditions. In particular, benzyl and dimethylaminophenyl esters yielded the corresponding dipeptides in several instances in high yields and with relatively small levels of hydrolysis. These results were also interpreted by computational docking analysis.
Cascade of youth? Resveratrol, the celebrated phytoalexin of red wine, was known to activate AMPK indirectly, but how this happened was unclear. In a paper recently published in Cell, S.-J. Park, J. H. Chung and co-workers identify the signalling cascade, which begins with the inhibition of phosphodiesterases, in particular PDE4. But questions remain, even while new perspectives open up.
We replaced the amino terminal Pro residue of the Plk1 polo-box-domain-binding pentapeptide (PLHSpT) with a library of N-alkyl-Gly “peptoids”, and identified long-chain tethered phenyl moieties giving greater than two-orders-of-magnitude affinity enhancement. Further simplification by replacing the peptoid residue with appropriate amides gave low-nanomolar affinity N-acylated tetrapeptides. Binding of the N-terminal long-chain phenyl extension was demonstrated by X-ray co-crystal data.Putting the hole in the polo: Replacing the amino terminal Pro residue of the Plk1 polo box domain (PBD)-binding pentamer peptide, PLHSpT, with a library of N-alkyl-Gly “peptoids” gave low-nanomolar affinity peptide–peptoid hybrids. An X-ray crystal structure of one PBD-bound hybrid showed that a long-chain alkylphenyl group created a hydrophobic channel (right) that was not present in the PLHSpT-bound complex (yellow circle, left).
The key to understanding: The application of expressed protein ligation and protein microarrays enabled an unparalleled insight into the complex interaction of phosphorylation and glycosylation on casein kinase 2 and its biological outcome.
Nonenzymatic DNA ligation chemistries containing a reversible step allow thermodynamic control of product formation, but they are not necessarily compatible with polymerase enzymes. We report a ligation system that uses commercially available reagents, includes a reversible step, and results in a linkage that can function as a template for PCR amplification with accurate sequence transfer.
Identifying baicalin-regulated genes for neuronal differentiation: Baicalin is a potent neuronal-differentiation-inducing compound. This study explored the gene expression regulated through baicalin-induced differentiation of C17.2 neural stem cells by using a DNA microarray followed by qPCR validation. The expression of 15 genes was significantly regulated among the 58 differentially expressed genes important for nervous system development and function.
Harvest time: Proflavine drives the reduction of NAD+ in the presence of a Rh-based electron mediator. Photoregenerated NADH was enzymatically active for oxidation by NADH-dependent L-glutamate dehydrogenase for the synthesis of L-glutamate. This work suggests that proflavine has the potential to become an efficient light-harvesting component in biocatalytic photosynthesis driven by solar energy.
Lantibiotics are a large group of ribosomally synthesized peptides post-translationally modified to incorporate the amino acid lanthionine. They are classified, according to their biosynthetic pathway and bioactivity, into three major subtypes. Of Actinomycetes type III lantibiotics, only four peptides (SapB, SapT, LabA1, and LabA2) have been described and structurally characterized, although homologous gene clusters are abundant in other Actinomycetes. All these gene clusters share a similar architecture with a characteristic Ser/Ser/Cys motif in precursor peptides, which has previously been suggested to act as a precursor for lanthionine (SapB) and labionin (LabA2) rings. Mass spectrometry screening led to the discovery and characterization of three new representatives of type III lantibiotics: Avermipeptin (Avi), Erythreapeptin (Ery), and Griseopeptin (Gri) from Streptomyces avermitilis DSM 46492, Saccharopolyspora erythraea NRRL 2338, and Streptomyces griseus DSM 40236, respectively. Apart from the assignment of these peptides to their corresponding gene clusters, additional investigations on Avi, Ery and Gri peptides indicate stepwise leader processing by putative aminopeptidase-like protease(s), thus yielding mixtures of differently N-terminal-processed lantibiotic peptides. Similar peptide processing was observed for a heterologously expressed eryth biosynthetic gene cluster expressed in a Streptomyces host system. Remarkably, all isolates of the new type III lantibiotics contain both the amino acids lanthionine and labionin, thus implying dual-mode cyclase activity of the processing lyase-kinase-cyclase enzymes. These findings have implications for the structures and maturation of other type III lantibiotics from Actinomycetes.Keeping in trim: MS screening has led to the discovery of three class III lantibiotics. Structural characterization by HPLC-ESI-MS/MS and GC-MS analyses suggests that these lantibiotics are a mixture of peptides containing both lanthionine and labionin as post-translational modifications. We also report nonspecific N-terminal proteolytic trimming of the precursor peptide, thereby yielding differently processed lantibiotics.
Unusually versatile: While the ?-carbon thioether linkage in lantibiotics has long been appreciated and is relatively well characterized, a recent publication shows that the unusual sulfur-to-?-carbon thioether crosslinks in subtilosin A are produced by a radical SAM enzyme, AlbA, that contains two [4?Fe4?S] clusters, thus highlighting the versatility of post-translational modifications in natural product biosynthesis.
Destroying aromaticity: A novel prenyltransferase (Trt2) involved in fungal meroterpenoid biosynthesis was shown to catalyze an unusual aromatic addition reaction onto a fully substituted aromatic ring. The prenylated product serves as a key intermediate in the biosynthesis of the most abundant series of meroterpenoids in fungi.
Mechanistic promiscuity: The (2-alkyl)-3-hydroxy-4(1H)-quinolone-cleaving dioxygenase Hod has an ?/?-hydrolase fold and a Ser/His/Asp triad in its active site. Isatoic anhydride, a suicide substrate of serine hydrolases, inactivates Hod by covalent modification of the active-site serine, thus indicating that the ?/?-hydrolase fold can accommodate dioxygenase chemistry without completely abandoning hydrolase-like properties.
Not without a chaperone: Pharmacological chaperones are designed to bind and ideally stabilise their target protein. Here, we elucidate the molecular mechanism of a potential pharmacological chaperone to treat phenylketonuria. The crystal structure of human phenylalanine hydroxylase with compound IV may help in the rational design of more efficient compounds to treat this disease.
The design, preparation and characterisation of a library of malachite green (MG) derivatives for two-photon RNA labelling is described. Some of these MG derivatives exhibit an increased affinity for an MG-aptamer, as well as improved two-photon sensitivity when compared to the classical malachite green chloride. The underlying mechanisms and potential benefits for in vivo RNA visualisation are discussed.A library of malachite green derivatives was designed in order to improve two-photon detection of a malachite green RNA aptamer. Some of the prepared derivatives show remarkable affinity for the aptamer and improved two-photon brightness as measured by two-photon fluorescence correlation spectroscopy. These new derivatives show lower toxicity than the classical malachite green oxalate.
Molecules that can target duplex DNA with sequence selectivity have the potential to be useful tools in genomic research and also as therapeutic agents. Homopurine–homopyrimidine stretches in duplex DNA can be recognized by homopurine or homopyrimidine TFOs (triplex-forming oligonucleotides) through the formation of triplex DNA. We have previously developed bicyclic nucleoside analogues (WNAs) for the formation of stable triplexes in the formation of stable antiparallel triplexes containing a TA or a CG interrupting site. In this study, we investigated the effects on triplex DNA formation of ortho-, meta-, and para-methyl substituent groups on the aromatic ring of the WNA analogue. It was found that the homopurine TFO containing meta- and para-methyl-substituted WNA-?T (mMe-WNA-?T, pMe-WNA-?T) stabilized triplexes containing a TA interrupting site or a GC site, respectively. Interestingly, the ortho-methyl-substituted WNA-?T (oMe-WNA-?T) efficiently promoted DNA strand displacement to form the TFO/pyrimidine duplex. A detailed investigation showed that the duplex was in the antiparallel orientation and that its formation took place prior to triplex formation with the need for a magnesium cation. NOESY measurements indicated a significant difference in the rotation flexibilities of the phenyl rings of WNA-?Ts: that is, the conformation of the ortho-methylated phenyl ring was stable in a temperature-independent manner. It was speculated that the initial formation of a ternary complex was followed by strand displacement and then the formation of the TFO/pyrimidine duplex together with the TFO2/pyrimidine triplex formation during the early stage, and that the equilibrium shifted to the triplex during the later stage. Although the detailed role is still uncertain, the fixed phenyl ring of oMe-WNA-?T might play a role in the displacement reaction.Strand invasion of duplex DNA: Bicyclic nucleoside analogues (WNAs) containing ortho-, meta-, and para-methyl-substituted aromatic rings were synthesized. Interestingly, the ortho-methyl-substituted WNA-?T (oMe-WNA-?T) efficiently promoted DNA strand displacement to form the TFO/pyrimidine duplex.
Egged on: Elaborate syntheses of biological macromolecules consisting of more than two different components is developing. Kajihara and co-workers have used a bio-resource to develop a new strategy for the semisynthesis of glycoproteins.
Protein methylation is catalyzed by S-adenosyl-L-methionine-dependent protein methyltransferases (MTases), and this posttranslational modification serves diverse cellular functions. Some MTases seem to exhibit broad substrate specificities and comprehensive methods for target profiling are needed. Here we report the synthesis of a new AdoMet analogue for enzymatic transfer of a small propargyl group and labeling of modified proteins through copper-catalyzed azide–alkyne cycloaddition (CuAAC). Replacement of sulfur by selenium strongly enhanced the stability of the progargylic cofactor, leading, in combination with better activation by the selenonium center, to higher enzymatic reactivity. A broad spectrum of wild-type protein MTases acting on lysine, arginine, and glutamine residues accept this cofactor and modified substrates can be efficiently labeled by CuAAC click chemistry.Element editing: S-Adenosyl-L-methionine-dependent methyltransferases can transfer extended chemical groups (such as the propargyl group, which combines smallness, activation, and the potential for labeling after transfer) from cofactor analogues containing unsaturated bonds next to the reactive carbon. Replacing the sulfur in an unstable propargylic analogue with selenium gave a cofactor suitable for efficient protein labeling.
PEGged out: Poly(ethylene glycol), a simple biocompatible polymer, can replace natural loop segments in a 56-residue protein domain with a well-defined tertiary structure. Biophysical characterization of chimeras of the protein GB1 coupled with molecular dynamics simulations show that PEG enhances local backbone torsional freedom without compromising the overall protein fold or function.
We have investigated the use of FlAsH, a small fluorogenic molecule that binds to tetracysteine motifs, to probe folding of the 15-HEAT repeat protein PR65A. PR65A is one of a special class of modular non-globular proteins known as tandem repeat proteins, which are composed of small structural motifs that stack to form elongated, one-dimensional architectures. We were able to introduce linear and bipartite tetracysteine motifs at several sites along the ?-helical HEAT array of PR65A without disrupting the structure or stability. When the linear tetracysteine motif CCPGCC was used, FlAsH fluorescence reported globally on the folding of the protein. When the tetracysteine motif was displayed in bipartite mode through the engineering of pairs of cysteines on adjacent HEAT repeats, FlAsH fluorescence became a reporter of local conformation and of oligomerisation. Thus, by designing FlAsH-binding sites at different locations along the repeat array one can interrogate specific properties of PR65A, paving the way for structure–function analysis of this protein both in vitro and in the cell.Local reporter: FlAsH, a small fluorogenic molecule that binds to tetracysteine motifs, was used to probe folding of the ?-helical 15-HEAT-repeat protein PR65A. When the tetracysteine motif was displayed in bipartite mode by using pairs of cysteines engineered on adjacent HEAT repeats, FlAsH was able to act as a reporter of local conformation and oligomerisation.
Add it and see it: The concept of “DNA traffic lights” for wavelength-shifting DNA probes has a great potential in the application of biosensors, for example, in DNA aptamers. A visual color change in the DNA aptasensor fluorescence from green to red occurs after specific target binding.
The natural product gambogic acid (GA) has shown significant potential as an anticancer agent as it is able to induce apoptosis in multiple tumor cell lines, including multidrug-resistant cell lines, as well as displaying antitumor activity in animal models. Despite the fact that GA has entered phase I clinical trials, the primary cellular target and mode of action of this compound remain unclear, although many proteins have been shown to be affected by it. By thorough analysis of several cellular organelles, at both the morphological and functional levels, we demonstrate that the primary effect of GA is at the mitochondria. We found that GA induces mitochondrial damage within minutes of incubation at low-micromolar concentrations. Moreover, a fluorescent derivative of GA was able to localize specifically to the mitochondria and was displaced from these organelles after competition with unlabeled GA. These findings indicate that GA directly targets the mitochondria to induce the intrinsic pathway of apoptosis, and thus represents a new member of the mitocans.Target acquired: Gambogic acid induces apoptosis by directly targeting mitochondria and represents a new class of mitocans. Using fluorescence microscopy and immunblotting, we show that apoptosis is induced by cleavage of caspase-3 and -9 and that induction of the mitochondrial pathway was not related to morphological changes to other subcellular structures.
CYP106A2 is known as a 15?-hydroxylase, but also shows minor 11?-hydroxylase activity for progesterone. 11?-Hydroxyprogesterone is an important pharmaceutical compound with anti-androgenic and blood-pressure-regulating activity. This work therefore focused on directing the regioselectivity of the enzyme towards hydroxylation at position 11 in the C ring of the steroid through a combination of saturation mutagenesis and rational site-directed mutagenesis. With the aid of data from a homology model of CYP106A2 containing docked progesterone, together with site-directed mutagenesis of active-site residues (Lisurek et al. ChemBioChem2008, 9, 1439–1449), a saturation mutagenesis library at positions A395 and G397 was created. Screening of the library identified the mutants A395I and A395W/G397K as having 11?-hydroxylase activities 8.9 and 11.5 times higher than that of the wild type (WT). In the next step, additional mutations were integrated by a rational site-directed mutagenesis approach to increase the catalytic efficiency. Of the 40 candidates analyzed, the mutants A106T/A395I, A106T/A395I/R409L, and T89N/A395I turned out to display increased 11?-hydroxylase selectivities and activities relative to the WT (14.3-, 12.6-, and 11.8-fold increases in selectivity and 39.3-, 108-, and 24.4- in kcat/Km). In the last step of the study, the best mutants were applied in a whole-cell biotransformation. In these experiments the production (percentage) of 15?-hydroxyprogesterone decreased from 50.4?% (wild type) to 4.8?% (mutant T89N/A395I), whereas that of 11?-hydroxyprogesterone increased from 27.7 to 80.9?%, thus demonstrating an impressive regioselectivity.Combination of diverse mutagenesis techniques leads to a significant change in the regioselectivity of CYP106A2-dependent progesterone hydroxylation. Application of the generated mutants in whole-cell bioconversions resulted in a decrease in 15?-hydroxyprogesterone production from 50.4?% (wild type) to 4.8?% (T89N/A395I mutant), whereas the formation of 11?-hydroxyprogesterone increased from 27.7 to 80.9?%.
IrIII–porphyrins are a relatively new group of phosphorescent dyes that have potential for oxygen sensing and labeling of biomolecules. The requirement of two axial ligands for the IrIII ion permits simple linkage of biomolecules by a one-step ligand-exchange reaction, for example, using precursor carbonyl chloride complexes and peptides containing histidine residue(s). Using this approach, we produced three complexes of IrIII–octaethylporphyrin with cell-penetrating (Ir1 and Ir2) and tumor-targeting (Ir3) peptides and studied their photophysical properties. All of the complexes were stable and possessed bright, long-decay (unquenched lifetimes exceeding 45 ?s) phosphorescence at around 650 nm, with moderate sensitivity to oxygen. The Ir1 and Ir2 complexes showed positive staining of a number of mammalian cell types, thus demonstrating localization similar to endoplasmic reticulum and ATP- and temperature-independent intracellular accumulation (direct translocation mechanism). Their low photo- and cytotoxicity allows intracellular oxygen to be probed.IrIII–octaethylporphyrins represent a new class of phosphorescent sensors for molecular oxygen (O2). The simple synthesis procedure is described for symmetric disubstitution with histidine-containing peptides, ?. A number of cell-penetrating adducts were prepared and evaluated for sensing intracellular O2 in mammalian cells.
MIMS visualises metabolism: A recent publication by Steinhauser and co-workers presents a novel application of multi-isotope mass spectrometry (MIMS) to visualise physiological metabolism in live mammalian organisms, and validate the “immortal strand hypothesis” of asymmetric chromosomal division of stem cells in the small intestine.
In order to evaluate the proposed biosynthetic pathway for the methylmannose (MMPs) polysaccharides produced by mycobacteria, two homologous series of synthetic ?-(1?4)-linked 3-O-methyl-mannopyranosides, one terminated at the non-reducing end by a free mannopyranose residue (unmethylated oligosaccharides; OS) and the other terminated by a 3-O-methyl-mannopyranose residue (methylated OS), were prepared and evaluated as potential acceptors of an ?-(1?4)-mannosyltransferase. Using a mycobacterial membrane preparation as the source of the transferase, it was found that unmethylated OS are better substrates for the enzyme compared to the methylated OS of the same length. These results are inconsistent with the proposed MMP biosynthetic pathway, which suggests only methylated OS are acceptors of this transferase. To confirm that the observed activity arose from the desired ?-(1?4)-mannosyltransferase, as opposed to other mannosyltransferases present in the membrane preparation, the products resulting from tetrasaccharides 4 (unmethylated OS) and 9 (methylated OS), which only differ in the terminal residue, were further analyzed. MALDI-MS, exo-glycosidase digestion and 1H NMR spectroscopy were used to evaluate the structures of these reaction products. These experiments revealed that the enzymatic products of both 4 and 9 contain only ?-(1?4)-linked mannose residues, confirming the activity of the ?-(1?4)-mannosyltransferase. This supports the finding that both methylated and unmethylated OS are acceptors of the enzyme. It was also demonstrated that a homologous series of oligosaccharides with different number of mannose residues were formed from both 4 and 9, as opposed to a single reaction product. These results, again, challenge the previously proposed MMP biosynthetic pathway involving alternating methylation and mannosylation reactions.Is methylation essential? Synthetic analogues of methylmannose polysaccharides (MMPs) were used to evaluate the substrate specificity of a mycobacterial ?-(1?4)-mannosyltransferase (ManT) involved in MMP biosynthesis. In contrast to the previous biosynthetic hypothesis that a methyl group on the non-reducing terminus is essential for ManT activity, our results suggest multiple mannosyl transfers precede methylation.
Milk and sugar? The elucidation of the catalytic mechanism and the development of the first known inhibitor for lacto-N-biosidases, which are important enzymes involved in the breakdown of human milk oligosaccharides, are described.
A versatile “clickable” nucleoside: Metabolic labeling of cells is useful in studying the dynamics of biological molecules. N6pA can be utilized by all three mammalian RNA polymerases, as well as poly(A) polymerase. Because of its alkyne modification, RNA labeled with N6pA can be visualized and purified by using click chemistry.
In the quest for the identification of catalytic transformations to be used in chemical biology and medicinal chemistry, we identified iron(III) meso-tetraarylporphines as efficient catalysts for the reduction of aromatic azides to their amines. The reaction uses thiols as reducing agents and tolerates water, air, and other biological components. A caged fluorophore was employed to demonstrate that the reduction can be performed even in living mammalian cells. However, in vivo experiments in nematodes (Caenorhabditis elegans) and zebrafish (Danio rerio) revealed a limitation to this method: the metabolic reduction of aromatic azides.A tolerant catalyst: The iron meso-tetraarylporphin-catalyzed reduction of aromatic azides with thiols is described and analyzed. The discovered system tolerates the presence of water, air, and other biological components, and can even be performed in living mammalian cells, thus providing a novel tool for signal amplification in molecular life sciences.
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