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α-Amylases are the workhorse enzymes of starch degradation. They are central to human health, including as targets for anti-diabetic compounds, but are also the key enzymes in the industrial processing of starch for biofuels, corn syrups, brewing and detergents. Dissection of the activity, specificity and stability of α-amylases is crucial to understanding their biology and allowing their exploitation. Yet, functional characterization lags behind DNA sequencing and genomics; and new tools are required for rapid analysis of α-amylase function. Here, we design, synthesize and apply new branched α-amylase activity-based probes. Using both α-1,6 branched and unbranched α-1,4 maltobiose activity-based probes we were able to explore the stability and substrate specificity of both a panel of human gut microbial α-amylases and a panel of industrially relevant α-amylases. We also demonstrate how we can detect and annotate the substrate specificity of α-amylases in the complex cell lysate of both a prominent gut microbe and a diverse compost sample by in-gel fluorescence and mass spectrometry. A toolbox of starch-active activity-based probes will enable rapid functional dissection of α-amylases. We envisage activity-based probes contributing to better selection and engineering of enzymes for industrial application as well as fundamental analysis of enzymes in human health., (© 2024 Wiley‐VCH GmbH.)

The human Golgi α-mannosidase, hGMII, removes two mannose residues from GlcNAc-Man 5 GlcNAc 2 to produce GlcNAcMan 3 GlcNAc 2 , the precursor of all complex N -glycans including tumour-associated ones. The natural product GMII inhibitor, swainsonine, blocks processing of cancer-associated N -glycans, but also inhibits the four other human α-mannosidases, rendering it unsuitable for clinical use. Our previous structure-guided screening of iminosugar pyrrolidine and piperidine fragments identified two micromolar hGMII inhibitors occupying the enzyme active pockets in adjacent, partially overlapping sites. Here we demonstrate that fusing these fragments yields swainsonine-configured indolizidines featuring a C3-substituent that act as selective hGMII inhibitors. Our structure-guided GMII-selective inhibitor design complements a recent combinatorial approach that yielded similarly configured and substituted indolizidine GMII inhibitors, and holds promise for the potential future development of anti-cancer agents targeting Golgi N -glycan processing.

The combined inhibition of endoplasmic reticulum (ER) α-glucosidases I and II has been shown to inhibit replication of a broad range of viruses that rely on ER protein quality control. We found, by screening a panel of deoxynojirimycin and cyclitol glycomimetics, that the mechanism-based ER α-glucosidase II inhibitor, 1,6- epi -cyclophellitol cyclosulfate, potently blocks SARS-CoV-2 replication in lung epithelial cells, halting intracellular generation of mature spike protein, reducing production of infectious progeny, and leading to reduced syncytium formation. Through activity-based protein profiling, we confirmed ER α-glucosidase II inhibition in primary airway epithelial cells, grown at the air-liquid interface. 1,6- epi -Cyclophellitol cyclosulfate inhibits early pandemic and more recent SARS-CoV-2 variants, as well as SARS-CoV and MERS-CoV. The reported antiviral activity is comparable to the best-in-class described glucosidase inhibitors, all competitive inhibitors also targeting ER α-glucosidase I and other glycoprocessing enzymes not involved in ER protein quality control. We propose selective blocking ER-resident α-glucosidase II in a covalent and irreversible manner as a new strategy in the search for effective antiviral agents targeting SARS-CoV-2 and other viruses that rely on ER protein quality control., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

Developing protein confinement platforms is an attractive research area that not only promotes protein delivery but also can result in artificial environment mimicking of the cellular one, impacting both the controlled release of proteins and the fundamental protein biophysics. Polymeric nanoparticles (PNPs) are attractive platforms to confine proteins due to their superior biocompatibility, low cytotoxicity, and controllable release under external stimuli. However, loading proteins into PNPs can be challenging due to the potential protein structural perturbation upon contacting the interior of PNPs. In this work, we developed a novel approach to encapsulate proteins in PNPs with the assistance of the zeolitic imidazolate framework (ZIF). Here, ZIF offers an additional protection layer to the target protein by forming the protein@ZIF composite via aqueous-phase cocrystallization. We demonstrated our platform using a model protein, lysozyme, and a widely studied PNP composed of poly(ethylene glycol)-poly(lactic- co -glycolic acid) (PEG-PLGA). A comprehensive study via standard loading and release tests as well as various spectroscopic techniques was carried out on lysozyme loaded onto PEG-PLGA with and without ZIF protection. As compared with the direct protein encapsulation, an additional layer with ZIF prior to loading offered enhanced loading capacity, reduced leaching, especially in the initial stage, led to slower release kinetics, and reduced secondary structural perturbation. Meanwhile, the function, cytotoxicity, and cellular uptake of proteins encapsulated within the ZIF-bound systems are decent. Our results demonstrated the use of ZIF in assisting in protein encapsulation in PNPs and established the basis for developing more sophisticated protein encapsulation platforms using a combination of materials of diverse molecular architectures and disciplines. As such, we anticipate that the protein-encapsulated ZIF systems will serve as future polymer protein confinement and delivery platforms for both fundamental biophysics and biochemistry research and biomedical applications where protein delivery is needed to support therapeutics and/or nutrients.

Biodegradation of insoluble biomass such as cellulose via carbohydrase enzymes is an effective approach to break down plant cell walls and extract valuable materials therein. Yet, the high cost and poor reusability of enzymes are practical concerns. We recently proved that immobilizing multiple digestive enzymes on metal-organic materials (MOMs) allows enzymes to be reused via gravimetric separation, improving the cost efficiency of cereal biomass degradation [ ACS Appl. Mater. Interfaces 2021 , 13 , 36, 43085-43093]. However, this strategy cannot be adapted for enzymes whose substrates or products are insoluble (e.g., cellulose crystals). Recently, we described an alternative approach based on magnetic metal-organic frameworks (MOFs) using model enzymes/substrates [ ACS Appl. Mater. Interfaces 2020 , 12 , 37, 41794-41801]. Here, we aim to prove the effectiveness of combining these two strategies in cellulose degradation. We immobilized multiple carbohydrase enzymes that cooperate in cellulose degradation via cocrystallization with Ca 2+ , a carboxylate ligand (BDC) in the absence and presence of magnetic nanoparticles (MNPs). We then compared the separation efficiency and enzyme reusability of the resultant multienzyme@Ca-BDC and multienzyme@MNP-Ca-BDC composites via gravimetric and magnetic separation, respectively, and found that, although both composites were effective in cellulose degradation in the first round, the multienzyme@MNP-Ca-BDC composites displayed significantly enhanced reusability. This work provides the first experimental demonstration of using magnetic solid supports to immobilize multiple carbohydrase enzymes simultaneously and degrade cellulose and promotes green/sustainable chemistry in three ways: (1) reusing the enzymes saves energy/sources to prepare them, (2) the synthetic conditions are "green" without generating unwanted wastes, and (3) using our composites to degrade cellulose is the first step of extracting valuable materials from sustainable biomasses such as plants whose growth does not rely on nonregeneratable resources.

Enzyme immobilization offers a number of advantages that improve biocatalysis; however, finding a proper way to immobilize enzymes is often a challenging task. Implanting enzymes in metal-organic frameworks (MOFs) via co-crystallization, also known as biomineralization, provides enhanced reusability and stability with minimal perturbation and substrate selectivity to the enzyme. Currently, there are limited metal-ligand combinations with a proper protocol guiding the experimental procedures. We have recently explored 10 combinations that allow custom immobilization of enzymes according to enzyme stability and activity in different metals/ligands. Here, as a follow-up of that work, we present a protocol for how to carry out custom immobilization of enzymes using the available combinations of metal ions and ligands. Detailed procedures to prepare metal ions, ligands, and enzymes for their co-crystallization, together with characterization and assessment, are discussed. Precautions for each experimental step and result analysis are highlighted as well. This protocol is important for enzyme immobilization in various research and industrial fields. Key features • A wide selection of metal ions and ligands allows for the immobilization of enzymes in metal-organic frameworks (MOFs) via co-crystallization. • Step-by-step enzyme immobilization procedure via co-crystallization of metal ions, organic linkers, and enzymes. • Practical considerations and experimental conditions to synthesize the enzyme@MOF biocomposites are discussed. • The demonstrated method can be generalized to immobilize other enzymes and find other metal ion/ligand combinations to form MOFs in water and host enzymes., Competing Interests: Competing interestsThe authors declare no competing interests., (©Copyright : © 2024 The Authors; This is an open access article under the CC BY-NC license.)

Siastatin B is a potent and effective iminosugar inhibitor of three diverse glycosidase classes, namely, sialidases, β- d -glucuronidases, and N -acetyl-glucosaminidases. The mode of inhibition of glucuronidases, in contrast to sialidases, has long been enigmatic as siastatin B appears too bulky and incorrectly substituted to be accommodated within a β- d -glucuronidase active site pocket. Herein, we show through crystallographic analysis of protein-inhibitor complexes that siastatin B generates both a hemiaminal and a 3-geminal diol iminosugar (3-GDI) that are, rather than the parent compound, directly responsible for enzyme inhibition. The hemiaminal product is the first observation of a natural product that belongs to the noeuromycin class of inhibitors. Additionally, the 3-GDI represents a new and potent class of the iminosugar glycosidase inhibitor. To substantiate our findings, we synthesized both the gluco - and galacto -configured 3-GDIs and characterized their binding both structurally and kinetically to exo-β- d -glucuronidases and the anticancer target human heparanase. This revealed submicromolar inhibition of exo-β- d -glucuronidases and an unprecedented binding mode by this new class of inhibitor. Our results reveal the mechanism by which siastatin B acts as a broad-spectrum glycosidase inhibitor, identify a new class of glycosidase inhibitor, and suggest new functionalities that can be incorporated into future generations of glycosidase inhibitors.