ChemBioChem最新文献

Clavulanic acid is a medicinally important inhibitor of serine β-lactamases (SBLs). We report studies on the mechanisms by which clavulanic acid inhibits representative Ambler class A (TEM-116), C (Escherichia coli AmpC), and D (OXA-10) SBLs using denaturing and non-denaturing mass spectrometry (MS). Similarly to observations with penam sulfones, most of the results support a mechanism involving acyl enzyme complex formation, followed by oxazolidine ring opening without efficient subsequent scaffold fragmentation (at pH 7.5). This observation contrasts with previous MS studies, which identified clavulanic acid scaffold fragmented species as the predominant SBL bound products. In all the SBLs studied here, fragmentation was promoted by acidic conditions, which are commonly used in LC‑MS analyses. Slow fragmentation was, however, observed under neutral conditions with TEM-116 on prolonged reaction with clavulanic acid. Although our results imply clavulanic acid scaffold fragmentation is likely not crucial for SBL inhibition in vivo, development of inhibitors that fragment to give stable covalent complexes is of interest.

Yellow fluorescent proteins (YFPs) are commonly used in biology to track cellular processes, particularly as acceptors in experiments using the Förster Resonant Energy Transfer (FRET) phenomenon. However, their fluorescence intensity is strongly pH-dependent, limiting their utility in acidic environments. Here, we explore the pH sensitivity of YFPs upon binding with an artificial repeat protein (αRep) both in vitro and in living cells. We show that αRep binds to Citrine, with high affinity in the nanomolar range at physiological and acidic pHs, leading to increased thermal stability of the complex. Moreover, αRep binding reduces Citrine's pKa by 0.75 pH units, leading to a decreased sensitivity to pH fluctuations. This effect can be generalized to other YFPs as Venus and EYFP in vitro. An efficient binding of αRep to Citrine has also been observed in living cells both at pH 7.4 and pH 6. This interaction leads to reduced variations of Citrine fluorescence intensity in response to pH variations in cells. Overall, the study highlights the potential of αReps as a tool to modulate the pH sensitivity of YFPs, paving the way for future exploration of biological events in acidic environments by FRET in combination with a pH-insensitive cyan donor.

Enzymatic platforms for producing malonyl-CoA-based extender units required for polyketide biosynthesis are often based on malonyl-CoA ligases such as MatB from Rhizobium trifolii and Rhodopseudomonas palustris. However, despite broad interest in the fluorination of polyketides and prior success with engineering MatB homologs, the suitability of MatB for accessing the tertiary substituted fluoromethylmalonyl-CoA needed to produce flurithromycin and solithromycin has not yet been reported. Herein, we report the structure-guided engineering of a MatB homolog to optimize the production of fluoromethylmalonyl-CoA, resulting in a variant with increased conversion and providing a platform to produce a suitable building block mixture for fluorinated macrolide production. Additionally, the mutant demonstrated broad utility for various substituted malonyl-CoAs. The MatB mutant sets the stage to access fluorinated macrolides by coupling it with altered PKS machinery to install fluorinated malonyl-CoA into macrolide scaffolds.

Indolocarbazoles are natural products with broad bioactivities. A distinct feature of indolocarbazole biosynthesis is the modification of the indole and maleimide rings by regioselective tailoring enzymes. Here, we study a new indolocarbazole variant, which is encoded by the acfXODCP genes from Streptomyces venezuelae ATCC 10712. First we characterise this pathway by expressing the acfXODCP genes in Streptomyces coelicolor, which led to the production of a C-5/C-5'-dihydroxylated indolocarbazole. We assign as a new product arcyriaflavin F. Second, we demonstrate the flavin-dependent monooxygenase AcfX catalyses the C-5/C-5' dihydroxylation of the unsubstituted arcyriaflavin A into arcyriaflavin F. Interestingly, AcfX shares homology to EspX from erdasporine A biosynthesis, which instead catalyses a single C-6 indolocarbazole hydroxylation. In summary, we report a new indolocarbazole biosynthetic pathway and a regioselective C-5 indole ring tailoring enzyme AcfX.

Nucleoside analogues are a promising class of natural compounds in the pharmaceutical industry, and many antiviral, antibacterial and anticancer drugs have been created through structural modification of nucleosides scaffold. Acyl protecting groups, especially the acetyl group, play an important role in the protection of hydroxy groups in nucleoside synthesis and modification; consequently, numerous methodologies have been put forth for the acetylation of free nucleosides. However, for nucleosides that contain different O- and N-based functionalities, selective deprotection of the acetyl group(s) in nucleosides has been studied little, despite its practical significance in simplifying the preparation of partially or differentially substituted nucleoside intermediates. In this mini-review, recent approaches for regioselective deacetylation in acetylated nucleosides and their analogues are summarized and evaluated. Different regioselectivities (primary ester, secondary ester, full de-O-acetylation, and de-N-acetylation) are summarized and discussed in each section.

Electron imaging of biological samples stained with heavy metals has enabled visualization of subcellular structures critical in chemical-, structural-, and neuro-biology. In particular, osmium tetroxide OsO4 has been widely adopted for selective lipid imaging. Despite the ubiquity of its use, the osmium speciation in lipid membranes and the process for contrast generation in electron microscopy (EM) have continued to be open questions, limiting efforts to improve staining protocols and therefore high-resolution nanoscale imaging of biological samples. Following our recent success using photoemission electron microscopy (PEEM) to image mouse brain tissues with synaptic resolution, we have used PEEM to determine the nanoscale electronic structure of Os-stained biological samples. Os(IV), in the form of OsO2, generates nanoaggregates in lipid membranes, leading to a strong spatial variation in the electronic structure and electron density of states. OsO2 has a metallic electronic structure that drastically increases the electron density of states near the Fermi level. Depositing metallic OsO2 in lipid membranes allows for strongly enhanced EM signals and conductivity of biological materials. The identification of the chemical species and understanding of the membrane contrast mechanism of Os-stained biological specimens provides a new opportunity for the development of staining protocols for high-resolution, high-contrast EM imaging.

Asymmetric hydrogenation of alkene moieties is important for the synthesis of chiral molecules, but achieving high stereoselectivity remains a challenge. Biocatalysis using ene-reductases (EReds) offers a viable solution. However, the need for NAD(P)H cofactors limits large-scale applications. Here, we explored an electrochemical alternative for recycling flavin-containing EReds using methyl viologen as a mediator. For this, we built a bio-electrocatalytic setup with an H-type glass reactor cell, proton exchange membrane, and carbon cloth electrode. Experimental results confirm the mediator's electrochemical reduction and enzymatic consumption. Optimization showed increased product concentration at longer reaction times with better reproducibility within 4-6 h. We tested two enzymes, Pentaerythritol Tetranitrate Reductase (PETNR) and the Thermostable Old Yellow Enzyme (TOYE), using different alkene substrates. TOYE showed higher productivity for the reduction of 2-cyclohexen-1-one (1.20 mM h-1), 2-methyl-2-cyclohexen-1-one (1.40 mM h-1) and 2-methyl-2-pentanal (0.40 mM h-1), with enantiomeric excesses ranging from 11% to 99%. PETNR outperformed TOYE in terms of enantioselectivity for the reduction of 2-methyl-2-pentanal (ee 59±7% (S)). Notably, TOYE achieved promising results also in reducing ketoisophorone, a challenging substrate, with similar enantiomeric excess compared to published values using NADH.

Cyanine-based near-infrared (NIR) fluorescent probes have played vital roles in biological application due to their low interference from background fluorescence, deep tissue penetration, high sensitivity, and minimal photodamage to biological samples. They are widely utilized in molecular recognition, medical diagnosis, biomolecular detection, and biological imaging. Herein, we provide a review of recent advancements in cyanine-based NIR fluorescent probes for the detection of pH, cells, tumor as well as their application in photothermal therapy (PTT) and photodynamic therapy (PDT).

Enzymatic degradation of polymers holds promise for advancing towards a bio-based economy. However, bulky polymers presents challenges in accessibility for biocatalysts, hindering depolymerization reactions. Beyond the impact of crystallinity, polymer chains can reside in different conformations affecting binding efficiency to the enzyme. We previously showed that the gauche and trans chain conformers associated with crystalline and amorphous regions of the synthetic polyethylene terephthalate (PET) display different affinity to PETase, thus affecting the depolymerization rate. However, structural-function relationships for biopolymers remain poorly understood in biocatalysis. In this study, we explored biodegradation of by-us previously synthesized bio-polyesters made from a rigid bicyclic chiral terpene-based diol and copolymerized with various renewable diesters. Herein, four of those polyesters spanning from semi-aromatic to aliphatic were subjected to enzymatic degradations in concert with induced-fit docking (IFD) analyses. Our findings demonstrate the importance of conformational selection in enzymatic depolymerization of biopolymers. A straight or twisted conformation of the polymer chain is crucial in biocatalytic degradation by showing different affinities to enzyme ground-state conformers. This work highlights the importance of considering the conformational match between the polymer and the enzyme to optimize the biocatalytic degradation efficiency of biopolymers, providing valuable insights for the development of sustainable bioprocesses.

Similar to ubiquitin, the ubiquitin-like protein NEDD8 is not only conjugated to other proteins but is itself subject to posttranslational modifications including lysine acetylation. Yet, compared to ubiquitin, only little is known about the biochemical and structural consequences of site-specific NEDD8 acetylation. Here, we generated site-specifically mono-acetylated NEDD8 variants for each known acetylation site by genetic code expansion. We show that, in particular, acetylation of K11 has a negative impact on the usage of NEDD8 by the NEDD8-conjugating enzymes UBE2M and UBE2F and that this is likely due to electrostatic and steric effects resulting in conformational changes of NEDD8. Finally, we provide evidence that p300 acts as a position-specific NEDD8 acetyltransferase.