[Fe]-hydrogenase contains the FeGP cofactor as the prosthetic group for activation of H₂. HcgA−G are involved in the biosynthesis of the cofactor. Most of the biosynthesis reactions were elucidated by structure-to-function analysis and in vitro biosynthesis. We describe the catalytic function of the Hcg proteins and the possibility of engineering of the FeGP cofactor in redesigned hydride-transfer enzymes.

Abstract

[Fe]-hydrogenase catalyzes the heterolytic cleavage of H₂ and reversible hydride transfer to methenyl-tetrahydromethanopterin. The iron-guanylylpyridinol (FeGP) cofactor is the prosthetic group of this enzyme, in which mononuclear Fe(II) is ligated with a pyridinol and two CO ligands. The pyridinol ligand fixes the iron by an acyl carbon and a pyridinol nitrogen. Biosynthetic proteins for this cofactor are encoded in the hmd co-occurring (hcg) genes. The function of HcgB, HcgC, HcgD, HcgE, and HcgF was studied by using structure-to-function analysis, which is based on the crystal structure of the proteins and subsequent enzyme assays. Recently, we reported the catalytic properties of HcgA and HcgG, novel radical S-adenosyl methionine enzymes, by using an in vitro biosynthesis assay. Here, we review the properties of [Fe]-hydrogenase and the FeGP cofactor, and the biosynthesis of the FeGP cofactor. Finally, we discuss the expected engineering of [Fe]-hydrogenase and the FeGP cofactor.

A practical and valuable all-chemical method has been developed to synthesize 5’-^7mGppp RNA on solid-support without damaging the ^7mG-cap or the RNA during RNA deprotection and release from the support under mild basic conditions. Substantial quantities of high-purity 5’-^7mGppp RNA are thus affordable as useful research tools.

Abstract

Given the importance of mRNA with 5’-cap, easy access to RNA substrates with different ^7mG caps, of high quality and in large quantities is essential to elucidate the roles of RNA and the regulation of underlying processes. In addition to existing synthetic routes to 5’-cap RNA based on enzymatic, chemical or chemo-enzymatic methods, we present here an all-chemical method for synthetic RNA capping. The novelty of this study lies in the fact that the capping reaction is performed on solid-support after automated RNA assembly using commercial 2’-O-propionyloxymethyl ribonucleoside phosphoramidites, which enable final RNA deprotection under mild conditions while preserving both ^7mG-cap and RNA integrity. The capping reaction is efficiently carried out between a 5’-phosphoroimidazolide RNA anchored on the support and ^7mGDP in DMF in the presence of zinc chloride. Substantial amounts of ^7mG-cap RNA (from 1 to 28 nucleotides in length and of any sequence with or without internal methylations) containing various cap structures (^7mGpppA, ^7mGpppA_m, ^7mGppp^m6A, ^7mGppp^m6A_m, ^7mGpppG, ^7mGpppG_m) were obtained with high purity after IEX-HPLC purification. This capping method using solid-phase chemistry is convenient to perform and provides access to valuable RNA substrates as useful research tools to unravel specific issues regarding cap-related processes.

The information on the potential therapeutic effects of terpenoids reviewed here should help to guide researchers in the development of safer and more effective drugs derived from natural terpenoid compounds for managing inflammatory problems.

Abstract

Human neutrophil elastase (HNE) is an enzyme that plays a key role in the body‘s inflammatory response. It has been linked to several diseases such as chronic obstructive pulmonary disease (COPD), emphysema, and cystic fibrosis. As potential treatments for these diseases, HNE inhibitors are of great interest. Metabolites derived from plants, particularly terpenoids such as β-caryophyllene found in black pepper and other plants, and geraniol present in several essential oils, are recognized as significant sources of inhibitors for HNE. Because of their ability to inhibit HNE, terpenoids are considered promising candidates for developing novel therapies to treat inflammatory conditions such as COPD and emphysema. Furthermore, nature can serve as an excellent designer, and it may offer a safer drug candidate for inhibiting HNE production and activity in the future. The Preferred Reporting Items for Systematic Reviews and Meta-Analyses were searched to get relevant and up-to-date literature on terpenoids as human neutrophil elastase inhibitors. This review focuses on the isolation, chemical diversity, and inhibition of human neutrophil elastase (HNE) of various terpenoids reported from natural sources up to 2022. A total of 251 compounds from various terpenoids classes have been reported. Further, it also provides a summary of HNE inhibitors and includes a thorough discussion on the structure-activity relationship.

MALT1 is a cysteine protease and the only human paracaspase. It is implicated in various human diseases. Here, an overview of the currently available molecular tools is given. Furthermore, their application and future possibilities are discussed. We expect that future research with these and improved tools will provide novel insights into MALT1’s pathobiological roles and enable use as diagnostic tools.

Abstract

The paracaspase MALT1 is a key regulator of the human immune response. It is implicated in a variety of human diseases. For example, deregulated protease activity drives the survival of malignant lymphomas and is involved in the pathophysiology of autoimmune/inflammatory diseases. Thus, MALT1 has attracted attention as promising drug target. Although many MALT1 inhibitors have been identified, molecular tools to study MALT1 activity, target engagement and inhibition in complex biological samples, such as living cells and patient material, are still scarce. Such tools are valuable to validate MALT1 as a drug target in vivo and to assess yet unknown biological roles of MALT1. In this review, we discuss the recent literature on the development and biological application of molecular tools to study MALT1 activity and inhibition.

Polyketides represent an area of significant interest for drug discovery efforts. Leveraging a combination of chemical synthesis and enzymology, researchers have routinely demonstrated that the best of both strategies are conferred and result in improved outcomes. This review highlights the success of such an approach and provides an outlook for polyketide synthetic biology.

Abstract

Polyketide natural products have significant promise as pharmaceutical targets for human health and as molecular tools to probe disease and complex biological systems. While the biosynthetic logic of polyketide synthases (PKS) is well-understood, biosynthesis of designer polyketides remains challenging due to several bottlenecks, including substrate specificity constraints, disrupted protein-protein interactions, and protein solubility and folding issues. Focusing on substrate specificity, PKSs are typically interrogated using synthetic thioesters. PKS assembly lines and their products offer a wealth of information when studied in a chemoenzymatic fashion. This review provides an overview of the past two decades of polyketide chemoenzymatic synthesis and their contributions to the field of chemical biology. These synthetic strategies have successfully yielded natural product derivatives while providing critical insights into enzymatic promiscuity and mechanistic activity.

Hypoxia in tumors alters cellular processes, affecting cancer cell behavior. Cobalt chloride-induced hypoxia reduced proliferation but increased migration and invasion in cancer cells. Short hypoxia increased galectin3 endocytosis, but prolonged hypoxia decreased it. Organelle changes indicated adaptation to hypoxic stress. Hypoxia modulates endocytic pathways, reducing proliferation and enhancing cell migration and invasion.

Abstract

Hypoxia, a decrease in cellular or tissue level oxygen content, is characteristic of most tumors and has been shown to drive cancer progression by altering multiple subcellular processes. We hypothesized that the cancer cells in a hypoxic environment might have slower proliferation rates and increased invasion and migration rates with altered endocytosis compared to the cancer cells in the periphery of the tumor mass that experience normoxic conditions. We induced cellular hypoxia by exposing cells to cobalt chloride, a chemical hypoxic mimicking agent. This study measured the effect of hypoxia on cell proliferation, migration, and invasion. Uptake of fluorescently labeled transferrin, galectin3, and dextran that undergo endocytosis through major endocytic pathways (Clathrin-mediated pathway (CME), Clathrin-independent pathway (CIE), Fluid phase endocytosis (FPE)) were analyzed during hypoxia. Also, the organelle changes associated with hypoxia were studied with organelle trackers. We found that the proliferation rate decreased, and the migration and invasion rate increased in cancer cells in hypoxic conditions compared to normoxic cancer cells. A short hypoxic exposure increased galectin3 uptake in hypoxic cancer cells, but a prolonged hypoxic exposure decreased clathrin-independent endocytic uptake of galectin 3. Subcellular organelles, such as mitochondria, increased to withstand the hypoxic stress, while other organelles, such as Endoplasmic reticulum (ER), were significantly decreased. These data suggest that hypoxia modulates cellular endocytic pathways with reduced proliferation and enhanced cell migration and invasion.

Rhomboid proteases are serine proteases that reside inside the lipid bilayer of a membrane. They are implicated in several diseases. We here report 4-oxo-β-lactam as a new scaffold for covalent rhomboid inhibitors and activity-based probes.

Abstract

Intramembrane serine proteases (rhomboid proteases) are involved in a variety of biological processes and are implicated in several diseases. Here, we report 4-oxo-β-lactams as a novel scaffold for inhibition of rhomboids. We show that they covalently react with the active site and that the covalent bond is sufficiently stable for detection of the covalent rhomboid-lactam complex. 4-Oxo-β-lactams may therefore find future use as both inhibitors and activity-based probes for rhomboid proteases.

We constructed a bifunctional fusion protein for FADH₂ regeneration and successfully coexpressed it with different flavin-dependent halogenases, as well as a dioxygenase that converts 6-chlorotryptophan to 4-Cl-Kynurenine. The figure is a still life of a laboratory bench, with a single oversized E. coli bacterium hovering over an Erlenmeyer flask. Cell disruption is depicted in visual analogy to cracking an egg with the lysate appearing like egg yolk. The lysate in the flask contains all necessary enzymes for the biocatalytic cascade described in the paper rendered as ribbon structures with coloring consistent with the paper. Shown as permanent marker notes on the bench surface are a key reaction scheme as well as a “ToDo-list” that checks off some important goals of the research work. More information can be found in the Research article by N. Montua, N. Sewald.

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.