Amino acid residues with opposite charges were introduced into the flexible region of IsPETase using a computation-based salt bridge design strategy. The mutation sites formed a salt bridge or salt bridge network to improve the thermal stability of IsPETase, and the degradation efficiency of amorphous PET film was enhanced.

Abstract

Polyethylene terephthalate (PET) is one of the most widely used plastics, and the accumulation of PET poses a great threat to the environment. IsPETase can degrade PET rapidly at moderate temperatures, but its application is greatly limited by the low stability. Herein, molecular dynamics (MD) simulations combined with a sequence alignment strategy were adopted to introduce salt bridges into the flexible region of IsPETase to improve its thermal stability. In the designed variants, the T _m values of IsPETase^I168R/S188D and IsPETase^I168R/S188E were 7.4 and 8.7 °C higher than that of the wild type, respectively. The release of products degraded by IsPETase^I168R/S188E was 4.3 times that of the wild type. Tertiary structure characterization demonstrated that the structure of the variants IsPETase^I168R/S188D and IsPETase^I168R/S188E became more compact. Extensive MD simulations verified that a stable salt bridge was formed between the residue R168 and D186 in IsPETase^I168R/S188D, while in IsPETase^I168R/S188E an R168-D186-E188 salt bridge network was observed. These results confirmed that the proposed computation-based salt bridge design strategy could efficiently generate variants with enhanced thermal stability for the long-term degradation of PET, which would be helpful for the design of enzymes with improved stability.

Drimane-type sesquiterpenes (DTSs) are bioactive natural products found in various organisms. We review the discovery of DTS synthases and the tailoring enzymes generating the chemical diversity. The catalytic motifs, domain functions and underlying mechanisms of the DTS synthases in constructing drimane scaffold are summarized. These discoveries present valuable opportunities for exploring DTSs biosynthesis through genome mining.

Abstract

Drimane-type sesquiterpenes (DTSs) are significant terpenoid natural products characterized by their unique C₁₅ bicyclic skeleton. They are produced by various organisms including plants, fungi, bacteria and marine organisms, and exhibit a diverse array of bioactivities. These bioactivities encompass antifeedant, anti-insecticidal, anti-bacterial, anti-fungal, anti-viral and anti-proliferative properties. Some DTSs contribute to the pungent flavor found in herb plants like water pepper, while others serve as active components responsible for the anti-cancer activities observed in medicinal mushrooms such as (−)-antrocin from Antrodia cinnamomea. Recently, DTS synthases have been identified in various organisms, biosynthesizing drimenol, drim-8-ene-11-ol and (+)-albicanol, which all possess the characteristic drimane skeleton. Interestingly, despite these enzymes producing chemical molecules with a drimane scaffold, they exhibit minimal amino acid sequence identity across different organisms. This Concept article focuses on the discovery of DTS synthases and the tailoring enzymes generating the chemical diversity of drimane natural products. We summarize and discuss their key features, including the chemical mechanisms, catalytic motifs and functional domains employed by these terpene synthases to generate DTS scaffolds.

A multi-enzyme cascade for the cell-free, one-pot synthesis of CDP-glycerol has been developed. Through a design-of-experiments approach, the yield of the cascade was increased from 10 to 89 % with respect to cytidine as a substrate. The final product titer after a batch time of 24 h was 31.2 mM CDP-glycerol.

Abstract

CDP-glycerol is a nucleotide-diphosphate-activated version of glycerol. In nature, it is required for the biosynthesis of teichoic acid in Gram-positive bacteria, which is an appealing target epitope for the development of new vaccines. Here, a cell-free multi-enzyme cascade was developed to synthetize nucleotide-activated glycerol from the inexpensive and readily available substrates cytidine and glycerol. The cascade comprises five recombinant enzymes expressed in Escherichia coli that were purified by immobilized metal affinity chromatography. As part of the cascade, ATP is regenerated in situ from polyphosphate to reduce synthesis costs. The enzymatic cascade was characterized at the laboratory scale, and the products were analyzed by high-performance anion-exchange chromatography (HPAEC)-UV and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). After the successful synthesis had been confirmed, a design-of-experiments approach was used to screen for optimal operation conditions (temperature, pH value and MgCl₂ concentration). Overall, a substrate conversion of 89 % was achieved with respect to the substrate cytidine.

A bifunctional HaloPROTAC ligand was developed to recruit the CRBN E3 ubiquitin ligase to degrade target proteins fused with a HaloTag in the cell. The CRBN-recruiting HaloPROTAC can expand the use of the HaloPROTAC system for screening the degradation targets for therapeutic discovery.

Abstract

Target validation is key to the development of protein degrading molecules such as proteolysis-targeting chimeras (PROTACs) to identify cellular proteins amenable for induced degradation by the ubiquitin-proteasome system (UPS). Previously the HaloPROTAC system was developed to screen targets of PROTACs by linking the chlorohexyl group with the ligands of E3 ubiquitin ligases VHL and cIAP1 to recruit target proteins fused to the HaloTag for E3-catalyzed ubiquitination. Reported here are HaloPROTACs that engage the cereblon (CRBN) E3 to ubiquitinate and degrade HaloTagged proteins. A focused library of CRBN-pairing HaloPROTACs was synthesized and screened to identify efficient degraders of EGFP-HaloTag fusion with higher activities than VHL-engaging HaloPROTACs at sub-micromolar concentrations of the compound. The CRBN-engaging HaloPROTACs broadens the scope of the E3 ubiquitin ligases that can be utilized to screen suitable targets for induced protein degradation in the cell.

The receptor activated by GLP-1 is a target for drugs that treat type 2 diabetes and obesity. Since glycine has a low α-helix propensity, and Gly22 of GLP-1 does not contact the receptor, we replaced Gly22 with residues that have a higher or lower helix propensity. Most substitutions had little effect on receptor affinity or activation and raised the possibility that the receptor-bound state of agonist peptides encompasses a broader conformational envelope.

Abstract

Agonists of the glucagon-like peptide-1 receptor (GLP-1R) are used to treat diabetes and obesity. Cryo-EM structures indicate that GLP-1 is completely α-helical when bound to the GLP-1R. The mature form of this hormone, GLP-1(7-36), contains a glycine residue near the center (Gly22). Since glycine has the second-lowest α-helix propensity among the proteinogenic α-amino acid residues, and Gly22 does not appear to make direct contact with the receptor, we were motivated to explore the impact on agonist activity of altering the α-helix propensity at this position. We examined GLP-1 analogues in which Gly22 was replaced with L-Ala, D-Ala, or β-amino acid residues with varying helix propensities. The results suggest that the receptor is reasonably tolerant of variations in helix propensity, and that the functional receptor-agonist complex may comprise a conformational spectrum rather than a single fixed structure.

Car crashes on highways require responses like rescuing victims, deviating traffic, towing away damaged vehicles, and sometimes even blocking a road. Similarly, during translation, ribosomes can collide into each other with potentially dangerous outcomes for the cell. In response to ribosome collisions, cells mount a multi-pronged response, activating several molecular ′squads′, symbolized by the Highway Police badge. These include degradation pathways for faulty mRNAs, peptide products and sometimes ribosomal constituents, regulation of translation, and stress as well as immune responses. More information can be found in the Review by M.-L. Winz et al.

We have synthesized perfluoroalkyl-containing amino acids (R_F-AA) and demonstrated that tripeptides composed of the R_F-AA have high cellular uptake efficiency. Furthermore, the absolute configuration of R_F-AA affected the uptake efficiency due to the change in its ability to form nanoparticles. We anticipate that these findings provide the foundation for the development of efficient cell-penetrating peptide (CPP). The cover picture shows tripeptides with perfluoroalkyl groups labeled with hydrophilic dyes that form particles in water and are efficiently taken up into cells. More information can be found in the Research Article by K. Aikawa et al.