Category Archives: ChemBioChem

Caulobacter segnis Dioxygenase CsO2: A Practical Biocatalyst for Stilbenoid Ozonolysis

Posted on 7 September 2023 by

The Caulobacter segnis dioxygenase has been employed for the enzymatic alkene cleavage of several stilbenoids, thus generating the corresponding aldehydes. CsO2-mediated ozonolysis represents a greener, safer, and more selective process with respect to conventional chemistry. To further assess the CsO2 usability as a preparative biocatalyst, a 50–mL scale biotransformation of resveratrol has been carried out obtaining the corresponding aldehydes with excellent yields (98 %), rapid reaction times (1.5 h) and unprecedented substrate-to-catalyst ratio (5 10³).

Abstract

Ozonolysis is a useful as well as dangerous reaction for performing alkene cleavage. On the other hand, enzymes are considered a more sustainable and safer alternative. Among them, Caulobacter segnis dioxygenase (CsO2) known so far for its ability to catalyze the coenzyme-free oxidation of vinylguaiacol into vanillin, was selected and its substrate scope evaluated towards diverse natural and synthetic stilbenoids. Under optimized conditions, CsO2 catalyzed the oxidative cleavage of the C=C double bonds of various trans-stilbenes, providing that a hydroxyl moiety was necessary in para-position of the phenyl group (e. g., resveratrol and its derivatives) for the reaction to take place, which was confirmed by modelling studies. The reactions occurred rapidly (0.5–3 h) with high conversions (95–99 %) and without formation of by-products. The resveratrol biotransformation was carried out on 50–mL scale thus confirming the feasibility of the biocatalytic system as a preparative method.

Structural Description of Chiral E‐Tiling DNA Nanotubes with the Chiral Indices (n,m) and Handedness Defined by Microscopic Imaging

Posted on 7 September 2023 by nFeiyang Feng, nShou‐Jun Xiaon

The chiral index theory, widely used in carbon nanotubes, has been introduced to describe the chiral structures of E-tiling DNA nanotubes precisely. In particular, we define a general equation of tube curvature with a clear physical picture by modifying its mathematical definition. Furthermore, we summarize the recent progress on defining the left- or right-handedness of E-tiling DNA nanotubes through differentiating their inside and outside surfaces by fluorescence or electron or atomic force microscopic imaging.

Abstract

In structural DNA nanotechnology, E-tiling DNA nanotubes are evidenced to be homogeneous in diameter and thus have great potential in biomedical applications such as cellular transport and communication, transmembrane ion/molecule channeling, and drug delivery. However, a precise structural description of chiral DNA nanotubes with chiral parameters was lacking, thus greatly hindering their application breadth and depth, until we recently raised and partly solved this problem. In this perspective, we summarize recent progress in defining the chiral indices and handedness of E-tiling DNA nanotubes by microscopic imaging, especially atomic force microscopy (AFM) imaging. Such a detailed understanding of the chiral structures of E-tiling DNA nanotubes will be very helpful in the future, on the one hand for engineering DNA nanostructures precisely, and, on the other, for realizing specific physicochemical properties and biological functions successfully.

The Role of Trp79 in β‐Actin on Histidine Methyltransferase SETD3 Catalysis

Posted on 5 September 2023 by

Second site: A distant Trp79 binding site modulates the histidine methyltransferase SETD3-catalysed methylation of His73 in β-actin. Our findings have implications for the identification of new substrates and chemical probes of biomedically important SETD3.

Abstract

N^τ-methylation of His73 in actin by histidine methyltransferase SETD3 plays an important role in stabilising actin filaments in eukaryotes. Mutations in actin and overexpression of SETD3 have been related to human diseases, including cancer. Here, we investigated the importance of Trp79 in β-actin on productive human SETD3 catalysis. Substitution of Trp79 in β-actin peptides by its chemically diverse analogues reveals that the hydrophobic Trp79 binding pocket modulates the catalytic activity of SETD3, and that retaining a bulky and hydrophobic amino acid at position 79 is important for efficient His73 methylation by SETD3. Molecular dynamics simulations show that the Trp79 binding pocket of SETD3 is ideally shaped to accommodate large and hydrophobic Trp79, contributing to the favourable release of water molecules upon binding. Our results demonstrate that the distant Trp79 binding site plays an important role in efficient SETD3 catalysis, contributing to the identification of new SETD3 substrates and the development of chemical probes targeting the biomedically important SETD3.

Exploring Photoswitchable Binding Interactions with Small‐Molecule‐ and Peptide‐Based Inhibitors of Trypsin

Posted on 4 September 2023 by

Different for different states: Small-molecule- and peptide-based photoswitchable inhibitors of trypsin were investigated to better understand their binding interactions and hence optimise the difference in biological activity between isomeric states. The best peptidic inhibitor displayed a more than fivefold difference in inhibitory activity between isomeric states compared to the best small-molecule inhibitor (3.4-fold), due to a more significant 3D structural change upon switching.

Abstract

The ability to photochemically activate a drug, both when and where needed, requires optimisation of the difference in biological activity between each isomeric state. As a step to this goal, we report small-molecule- and peptide-based inhibitors of the same protease—trypsin—to better understand how photoswitchable drugs interact with their biological target. The best peptidic inhibitor displayed a more than fivefold difference in inhibitory activity between isomeric states, whereas the best small-molecule inhibitor only showed a 3.4-fold difference. Docking and molecular modelling suggest this result is due to a large change in 3D structure in the key binding residues of the peptidic inhibitor upon isomerisation; this is not observed for the small-molecule inhibitor. Hence, we demonstrate that significant structural changes in critical binding motifs upon irradiation are essential for maximising the difference in biological activity between isomeric states. This is an important consideration in the design of future photoswitchable drugs for clinical applications.

Chemo‐Enzymatic Fluorescence Labeling Of Genomic DNA For Simultaneous Detection Of Global 5‐Methylcytosine And 5‐Hydroxymethylcytosine

Posted on 4 September 2023 by

Dual-color global labelling of 5-hydroxymethylcytosine and umCpG by multi-color fluorescent labelling. We apply an engineered methyltransferase enzyme specific for unmodified CpG to incorporate a modified cofactor that binds to a fluorophore by click chemistry. In combination with 5-hydroxymethylcytosine labelling via enzymatic glycosylation, we incorporate spectrally distinct colour for each epigenetic mark, enabling simultaneous quantification in different cancer types.

Abstract

5-Methylcytosine and 5-hydroxymethylcytosine are epigenetic modifications involved in gene regulation and cancer. We present a new, simple, and high-throughput platform for multi-color epigenetic analysis. The novelty of our approach is the ability to multiplex methylation and de-methylation signals in the same assay. We utilize an engineered methyltransferase enzyme that recognizes and labels all unmodified CpG sites with a fluorescent cofactor. In combination with the already established labeling of the de-methylation mark 5-hydroxymethylcytosine via enzymatic glycosylation, we obtained a robust platform for simultaneous epigenetic analysis of these marks. We assessed the global epigenetic levels in multiple samples of colorectal cancer and observed a 3.5-fold reduction in 5hmC levels but no change in DNA methylation levels between sick and healthy individuals. We also measured epigenetic modifications in chronic lymphocytic leukemia and observed a decrease in both modification levels (5-hydroxymethylcytosine: whole blood 30 %; peripheral blood mononuclear cells (PBMCs) 40 %. 5-methylcytosine: whole blood 53 %; PBMCs 48 %). Our findings propose using a simple blood test as a viable method for analysis, simplifying sample handling in diagnostics. Importantly, our results highlight the assay‘s potential for epigenetic evaluation of clinical samples, benefiting research and patient management.

Hydrogels with Reversible Crosslinks for Improved Localised Stem Cell Retention: A Review

Posted on 29 August 2023 by

Tissue repair by administering stem cells retained in adaptable hydrogels. Various promising stem cells for tissue repair are discussed, together with the hydrogels that enclose them and interact well with the tissue. Hydrogel–cell fixation is accomplished by the hydrogel's chemical design, and the injectability by shear-thinning. Due to the dynamic bonds, the hydrogel will become injectable by applying force, but form a gel after injection.

Abstract

Successful stem cell applications could have a significant impact on the medical field, where many lives are at stake. However, the translation of stem cells to the clinic could be improved by overcoming challenges in stem cell transplantation and in vivo retention at the site of tissue damage. This review aims to showcase the most recent insights into developing hydrogels that can deliver, retain, and accommodate stem cells for tissue repair. Hydrogels can be used for tissue engineering, as their flexibility and water content makes them excellent substitutes for the native extracellular matrix. Moreover, the mechanical properties of hydrogels are highly tuneable, and recognition moieties to control cell behaviour and fate can quickly be introduced. This review covers the parameters necessary for the physicochemical design of adaptable hydrogels, the variety of (bio)materials that can be used in such hydrogels, their application in stem cell delivery and some recently developed chemistries for reversible crosslinking. Implementing physical and dynamic covalent chemistry has resulted in adaptable hydrogels that can mimic the dynamic nature of the extracellular matrix.

Activity‐Based Protein Profiling in Methicillin‐Resistant Staphylococcus aureus Reveals the Broad Reactivity of a Carmofur‐Derived Probe

Posted on 29 August 2023 by nMd Jalal Uddin, nHermen S. Overkleeft, nChristian S. Lentzn

Enzyme targets of activity-based probes built on the anti-neoplastic drug and antimicrobial agent carmofur in methicillin-resistant Staphylococcus aureus (MRSA) have been identified. Chemoproteomic profiling revealed a broad reactivity of the probe against both cysteine and serine hydrolases, and identified uncharacterized bacterial enzymes; this has implications for putative human or microbial (off)targets engaged by therapeutic use of carmofur in the clinic.

Abstract

Activity-based protein profiling is a powerful chemoproteomic technique to detect active enzymes and identify targets and off-targets of drugs. Here, we report the use of carmofur- and activity-based probes to identify biologically relevant enzymes in the bacterial pathogen Staphylococcus aureus. Carmofur is an anti-neoplastic prodrug of 5-fluorouracil and also has antimicrobial and anti-biofilm activity. Carmofur probes were originally designed to target human acid ceramidase, a member of the NTN hydrolase family with an active-site cysteine nucleophile. Here, we first profiled the targets of a fluorescent carmofur probe in live S. aureus under biofilm-promoting conditions and in liquid culture, before proceeding to target identification by liquid chromatography/mass spectrometry. Treatment with a carmofur-biotin probe led to enrichment of 20 enzymes from diverse families awaiting further characterization, including the NTN hydrolase-related IMP cyclohydrolase PurH. However, the probe preferentially labeled serine hydrolases, thus displaying a reactivity profile similar to that of carbamates. Our results suggest that the electrophilic N-carbamoyl-5-fluorouracil scaffold could potentially be optimized to achieve selectivity towards diverse enzyme families. The observed promiscuous reactivity profile suggests that the clinical use of carmofur presumably leads to inactivation of a number human and microbial enzymes, which could lead to side effects and/or contribute to therapeutic efficacy.

CuII Pyrazolyl‐Benzimidazole Dinuclear Complexes with Remarkable Antioxidant Activity

Posted on 29 August 2023 by

The novel dinuclear Cu^II complex [Cu(NO₃)(μ-NO₃)(L^’)]₂ , containing an oxidized ligand, shows remarkable antioxidant properties compared to the original ligand and reference compounds. A mechanism is provided.

Abstract

Three dinuclear coordination complexes generated from 1-n-butyl-2-((5-methyl-1H-pyrazole-3-yl)methyl)-1H-benzimidazole (L), have been synthesized and characterized spectroscopically and structurally by single crystal X-ray diffraction analysis. Reaction with iron(II) chloride and then copper(II) nitrate led to a co-crystal containing 78 % of [Cu(NO₃)(μ-Cl)(L’)]₂ (C₁ ) and 22 % of [Cu(NO₃)(μ-NO₃)(L’)]₂ (C₂ ), where L was oxidized to a new ligand L^’ . A mechanism is provided. Reaction with copper chloride led to the dinuclear complex [Cu(Cl)(μ-Cl)(L)]₂ (C₃ ). The presence of N−H⋅⋅⋅O and C−H⋅⋅⋅O intermolecular interactions in the crystal structure of C₁ and C₂ , and C−H⋅⋅⋅N and C−H⋅⋅⋅Cl hydrogen bonding in the crystal structure of C₃ led to supramolecular structures that were confirmed by Hirshfeld surface analysis. The ligands and their complexes were tested for free radical scavenging activity and ferric reducing antioxidant power. The complex C₁ /C₂ shows remarkable antioxidant activities as compared to the ligand L and reference compounds.

Extended Biocatalytic Halogenation Cascades Involving a Single‐Polypeptide Regeneration System for Diffusible FADH2

Posted on 29 August 2023 by nNicolai Montua, nNorbert Sewaldn

A bifunctional fusion enzyme with phosphite dehydrogenase and flavin reductase activities has been constructed and characterised. Co-expression of this single polypeptide regeneration system with tryptophan halogenases and carrier-free immobilisation in combiCLEAs facilitates preparative-scale synthesis of halotryptophan from a single cultivation. Extension of the catalytic cascade with a specific dioxygenase enables single cultivation one-pot synthesis of l-4-Cl-kynurenine on a preparative scale.

Abstract

Flavin-dependent halogenases have attracted increasing interest for aryl halogenation at unactivated C−H positions because they are characterised by high regioselectivity, while requiring only FADH₂, halide salts, and O₂. Their use in combined crosslinked enzyme aggregates (combiCLEAs) together with an NADH-dependent flavin reductase and an NADH-regeneration system for the preparative halogenation of tryptophan and indole derivatives has been previously described. However, multiple cultivations and protein purification steps are necessary for their production. We present a bifunctional regeneration enzyme for two-step catalytic flavin regeneration using phosphite as an inexpensive sacrificial substrate. This fusion protein proved amenable to co-expression with various flavin-dependent Trp-halogenases and enables carrier-free immobilisation as combiCLEAs from a single cultivation for protein production and the preparative synthesis of halotryptophan. The scalability of this system was demonstrated by fed-batch fermentation in bench-top bioreactors on a 2.5 L scale. Furthermore, the inclusion of a 6-halotryptophan-specific dioxygenase into the co-expression strain further converts the halogenation product to the kynurenine derivative. This reaction cascade enables the one-pot synthesis of l-4-Cl-kynurenine and its brominated analogue on a preparative scale.

Bacterial J‐Domains with C‐Terminal Tags Contact the Substrate Binding Domain of DnaK and Sequester Chaperone Activity

Posted on 29 August 2023 by nBrock Nelson, nNathan Soper, nTania J. Lupolin

J-proteins are cofactors that assist molecular chaperones in cellular protein folding. We have found that short C-terminal tags added to the N-terminal domains of J-proteins (J-domains) can promote interactions with multiple domains of DnaK, including the substrate binding domain. These modified J-domains are capable of disrupting DnaK chaperone activity in vitro, and recovery following proteotoxic stress in cells. This work highlights the importance of C-terminal sequences in J-protein function.

Abstract

Functional interactions between the molecular chaperone DnaK and cofactor J-proteins (DnaJs), as well as their homologs, are crucial to the maintenance of proteostasis across cell types. In the bacterial pathogen Mycobacterium tuberculosis, DnaK–DnaJ interactions are essential for cell growth and represent potential targets for antibiotic or adjuvant development. While the N-terminal J-domains of J-proteins are known to form important contacts with DnaK, C-terminal domains have varied roles. Here, we have studied the effect of adding C-terminal tags to N-terminal J-domain truncations of mycobacterial DnaJ1 and DnaJ2 to promote additional interactions with DnaK. We found that His₆ tags uniquely promote binding to additional sites in the substrate binding domain at the C-terminus of DnaK. Other C-terminal tags attached to J-domains, even peptides known to interact with DnaK, do not produce the same effects. Expression of C-terminally modified DnaJ1 or DnaJ2 J-domains in mycobacterial cells suppresses chaperone activity following proteotoxic stress, which is exaggerated in the presence of a small-molecule DnaK inhibitor. Hence, this work uncovers genetically encodable J-protein variants that may be used to study chaperone–cofactor interactions in other organisms.