Search Results (18)

Carboxypeptidase is a Zn-dependent protease that specifically recognises and hydrolyses peptides with a hydrophobic side chain at the C-terminal residue. According to hydrolysis mechanisms proposed in the literature, catalysis requires a water molecule to be close to the Zn ion so as to be activated as a nucleophile. Among small molecules that resemble the slowly hydrolysed Gly-Tyr peptide, which have been previously designed as inhibitors and characterised structurally, a variant with the terminal amino acid in a D-configuration has been the most effective. Our molecular dynamics simulations of carboxypeptidase complexed with different variants of those inhibitor ligands as well as variants of the Gly-Tyr peptide show that the strength of the inhibitory effect is not related to the binding strength of the ligand. Our data rather support an earlier notion that the inhibition is, at least partially, due to blocking a coordination site at the Zn ion by the ligand coordinating the metal ion in a bidentate fashion. Full article

The SARS-CoV-2 main protease (M^pro) is initially synthesized as part of polyprotein precursors that undergo autoproteolysis to release the free mature M^pro. To investigate the autoprocessing mechanism in transfected mammalian cells, we examined several fusion precursors, with the mature SARS-CoV-2 M^pro along with the flanking amino acids (to keep the native substrate sequences) sandwiched between different tags. Our analyses revealed differential proteolysis kinetics at the N- and C-terminal cleavage sites. Particularly, N-terminal processing is differentially influenced by various upstream fusion tags (GST, sGST, CD63, and Nsp4) and amino acid variations at the N-terminal P1 position, suggesting that precursor catalysis is flexible and subject to complex regulation. Mutating Q to E at the N-terminal P1 position altered both precursor catalysis and the properties of the released M^pro. Interestingly, the wild-type precursors exhibited different enzymatic activities compared to those of the released M^pro, displaying much lower susceptibility to known inhibitors targeting the mature form. These findings suggest the precursors as alternative targets for antiviral development. Accordingly, we developed and validated a high-throughput screening (HTS)-compatible platform for functional screening of compounds targeting either the N-terminal processing of the SARS-CoV-2 M^pro precursor autoprocessing or the released mature M^pro through different mechanisms of action. Full article

Proteases represent common targets in combating infectious diseases, including COVID-19. The 3-chymotrypsin-like protease (3CLpro) is a validated molecular target for COVID-19, and it is key for developing potent and selective inhibitors for inhibiting viral replication of SARS-CoV-2. In this review, we discuss structural relationships and diverse subsites of 3CLpro, shedding light on the pivotal role of dimerization and active site architecture in substrate recognition and catalysis. Our analysis of bioinformatics and other published studies motivated us to investigate a novel catalytic mechanism for the SARS-CoV-2 polyprotein cleavage by 3CLpro, centering on the triad mechanism involving His41-Cys145-Asp187 and its indispensable role in viral replication. Our hypothesis is that Asp187 may participate in modulating the pK_a of the His41, in which catalytic histidine may act as an acid and/or a base in the catalytic mechanism. Recognizing Asp187 as a crucial component in the catalytic process underscores its significance as a fundamental pharmacophoric element in drug design. Next, we provide an overview of both covalent and non-covalent inhibitors, elucidating advancements in drug development observed in preclinical and clinical trials. By highlighting various chemical classes and their pharmacokinetic profiles, our review aims to guide future research directions toward the development of highly selective inhibitors, underscore the significance of 3CLpro as a validated therapeutic target, and propel the progression of drug candidates through preclinical and clinical phases. Full article

Microbial degradation of feathers offers potential for bioremediation, yet the microbial response mechanisms warrant additional investigation. In prior work, Pseudomonas aeruginosa Gxun-7, which demonstrated robust degradation of feathers at elevated concentrations, was isolated. However, the molecular mechanism of this degradation remains only partially understood. To investigate this, we used RNA sequencing (RNA-seq) to examine the genes that were expressed differentially in P. aeruginosa Gxun-7 when exposed to 25 g/L of feather substrate. The RNA-seq analysis identified 5571 differentially expressed genes; of these, 795 were upregulated and 603 were downregulated. Upregulated genes primarily participated in proteolysis, amino acid, and pyruvate metabolism. Genes encoding proteases, as well as those involved in sulfur metabolism, phenazine synthesis, and type VI secretion systems, were notably elevated, highlighting their crucial function in feather decomposition. Integration of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) taxonomies, combined with a review of the literature, led us to propose that metabolic feather degradation involves environmental activation, reducing agent secretion, protease release, peptide/amino acid uptake, and metabolic processes. Sulfite has emerged as a critical activator of keratinase catalysis, while cysteine serves as a regulatory mediator. qRT–PCR assay results for 11 selected gene subset corroborated the RNA-seq findings. This study enhances our understanding of the transcriptomic responses of P. aeruginosa Gxun-7 to feather degradation and offers insights into potential degradation mechanisms, thereby aiding in the formulation of effective feather waste management strategies in poultry farming. Full article

Proteins that use cysteine residues for catalysis or regulation are widely distributed and intensively studied, with many biomedically important examples. Enzymes where cysteine is a catalytic nucleophile typically generate covalent catalytic intermediates whose structures are important for understanding mechanism and for designing targeted inhibitors. The formation of catalytic intermediates can change enzyme conformational dynamics, sometimes activating protein motions that are important for catalytic turnover. However, these transiently populated intermediate species have been challenging to structurally characterize using traditional crystallographic approaches. This review describes the use and promise of new time-resolved serial crystallographic methods to study cysteine-dependent enzymes, with a focus on the main (M^pro) and papain-like (PL^pro) cysteine proteases of SARS-CoV-2, as well as on other examples. We review features of cysteine chemistry that are relevant for the design and execution of time-resolved serial crystallography experiments. In addition, we discuss emerging X-ray techniques, such as time-resolved sulfur X-ray spectroscopy, that may be able to detect changes in sulfur charge states and covalency during catalysis or regulatory modification. In summary, cysteine-dependent enzymes have features that make them especially attractive targets for new time-resolved serial crystallography approaches, which can reveal both changes to enzyme structures and dynamics during catalysis in crystalline samples. Full article

5-hydroxymethylfurfural (HMF) is a platform chemical that can be converted into a wide range of high-value derivatives. Industrially, HMF-based derivatives are synthesized via chemical catalysis. However, biocatalytic transformation has emerged as an attractive alternative. Significant advances have been made in the last years using isolated enzymes and whole-cell biocatalysts in HMF biotransformation. Nonetheless, one of the major bottlenecks is the cost of the process, mainly due to the microorganism growth substrate. In this work, biotransformation studies to transform HMF into 2,5-di(hydroxymethyl)furan (DHMF) were carried out with the fungus Fusarium striatum using low-cost protein hydrolysates. The protein hydrolysates were obtained from fines, an unexploited material produced during the rendering process of meat industry waste residues. Given the high content in the protein of fines, of around 46%, protein hydrolysis was optimized using two commercially available proteases, Alcalase 2.4 L and Neutrase 0.8 L. The maximum degree of hydrolysis (DH) achieved with Alcalase 2.4 L was 21.4% under optimal conditions of 5% E/S ratio, pH 8, 55 °C, and 24 h. On the other hand, Neutrase 0.8 L exhibited lower efficiency, and therefore, lower protein recovery. After optimization of the Neutrase 0.8 L process using the response surface methodology (RSM), the maximum DH achieved was 7.2% with the variables set at 15% E/S ratio, initial pH 8, 40 °C, and 10.5 h. Using these hydrolysates as a nitrogen source allowed higher sporulation of the fungus and, therefore, the use of a lower volume of inoculum (three-fold), obtaining a DHMF yield > 90%, 50% higher than the yield obtained when using commercial peptones. The presented process allows the transformation of animal co- and by-products into low-cost nitrogen sources, which greatly impacts the industrial feasibility of HMF biotransformation. Full article

A growing number of studies indicate that mRNAs and long ncRNAs can affect protein populations by assembling dynamic ribonucleoprotein (RNP) granules. These phase-separated molecular ‘sponges’, stabilized by quinary (transient and weak) interactions, control proteins involved in numerous biological functions. Retroviruses such as HIV-1 form by self-assembly when their genomic RNA (gRNA) traps Gag and GagPol polyprotein precursors. Infectivity requires extracellular budding of the particle followed by maturation, an ordered processing of ∼2400 Gag and ∼120 GagPol by the viral protease (PR). This leads to a condensed gRNA-NCp7 nucleocapsid and a CAp24-self-assembled capsid surrounding the RNP. The choreography by which all of these components dynamically interact during virus maturation is one of the missing milestones to fully depict the HIV life cycle. Here, we describe how HIV-1 has evolved a dynamic RNP granule with successive weak–strong–moderate quinary NC-gRNA networks during the sequential processing of the GagNC domain. We also reveal two palindromic RNA-binding triads on NC, KxxFxxQ and QxxFxxK, that provide quinary NC-gRNA interactions. Consequently, the nucleocapsid complex appears properly aggregated for capsid reassembly and reverse transcription, mandatory processes for viral infectivity. We show that PR is sequestered within this RNP and drives its maturation/condensation within minutes, this process being most effective at the end of budding. We anticipate such findings will stimulate further investigations of quinary interactions and emergent mechanisms in crowded environments throughout the wide and growing array of RNP granules. Full article

The astacin protease Meprin β represents an emerging target for drug development due to its potential involvement in disorders such as acute and chronic kidney injury and fibrosis. Here, we elaborate on the structural basis of inhibition by a specific Meprin β inhibitor. Our analysis of the crystal structure suggests different binding modes of the inhibitor to the active site. This flexibility is caused, at least in part, by movement of the C-terminal region of the protease domain (CTD). The CTD movement narrows the active site cleft upon inhibitor binding. Compared with other astacin proteases, among these the highly homologous isoenzyme Meprin α, differences in the subsites account for the unique selectivity of the inhibitor. Although the inhibitor shows substantial flexibility in orientation within the active site, the structural data as well as binding analyses, including molecular dynamics simulations, support a contribution of electrostatic interactions, presumably by arginine residues, to binding and specificity. Collectively, the results presented here and previously support an induced fit and substantial movement of the CTD upon ligand binding and, possibly, during catalysis. To the best of our knowledge, we here present the first structure of a Meprin β holoenzyme containing a zinc ion and a specific inhibitor bound to the active site. The structural data will guide rational drug design and the discovery of highly potent Meprin inhibitors. Full article

Inquiries into the participation of short hydrogen bonds in stabilizing transition states and intermediate states in the thrombin, factor Xa, plasmin and activated protein C–catalyzed reactions revealed that specific binding of effectors at S_n, n = 1–4 and S’_n, n = 1–3 and at remote exosites elicit complex patterns of hydrogen bonding and involve water networks. The methods employed that yielded these discoveries include; (1) kinetics, especially partial or full kinetic deuterium solvent isotope effects with short cognate substrates and also with the natural substrates, (2) kinetic and structural probes, particularly low-field high-resolution nuclear magnetic resonance (¹H NMR), of mechanism-based inhibitors and substrate-mimic peptide inhibitors. Short hydrogen bonds form at the transition states of the catalytic reactions at the active site of the enzymes as they do with mechanism-based covalent inhibitors of thrombin. The emergence of short hydrogen bonds at the binding interface of effectors and thrombin at remote exosites has recently gained recognition. Herein, I describe our contribution, a confirmation of this discovery, by low-field ¹H NMR. The principal conclusion of this review is that proton sharing at distances below the sum of van der Waals radii of the hydrogen and both donor and acceptor atoms contribute to the remarkable catalytic prowess of serine proteases of the blood clotting system and other enzymes that employ acid-base catalysis. Proton bridges also play a role in tight binding in proteins and at exosites, i.e., allosteric sites, of enzymes. Full article

Significance: The majority of the drugs which target membrane-embedded protease γ-secretase show an unusual biphasic activation–inhibition dose-response in cells, model animals, and humans. Semagacestat and avagacestat are two biphasic drugs that can facilitate cognitive decline in patients with Alzheimer’s disease. Initial mechanistic studies showed that the biphasic drugs, and pathogenic mutations, can produce the same type of changes in γ-secretase activity. Results: DAPT, semagacestat LY-411,575, and avagacestat are four drugs that show different binding constants, and a biphasic activation–inhibition dose-response for amyloid-β-40 products in SH-SY5 cells. Multiscale molecular dynamics studies have shown that all four drugs bind to the most mobile parts in the presenilin structure, at different ends of the 29 Å long active site tunnel. The biphasic dose-response assays are a result of the modulation of γ-secretase activity by the concurrent binding of multiple drug molecules at each end of the active site tunnel. The drugs activate γ-secretase by facilitating the opening of the active site tunnel, when the rate-limiting step is the tunnel opening, and the formation of the enzyme–substrate complex. The drugs inhibit γ-secretase as uncompetitive inhibitors by binding next to the substrate, to dynamic enzyme structures which regulate processive catalysis. The drugs can modulate the production of different amyloid-β catalytic intermediates by penetration into the active site tunnel, to different depths, with different flexibility and different binding affinity. Conclusions: Biphasic drugs and pathogenic mutations can affect the same dynamic protein structures that control processive catalysis. Successful drug-design strategies must incorporate transient changes in the γ-secretase structure in the development of specific modulators of its catalytic activity. Full article

The recovery of the protein component and its transformation into protein hydrolysates, generally carried out chemically, gives great added value to waste biomasses. The production of protein hydrolysates through enzymatic catalysis would guarantee to lower the environmental impact of the process and raise product quality, due to the reproducible formation of low molecular weight peptides, with interesting and often unexplored biological activities. The immobilization of the enzymes represents a good choice in terms of stability, recyclability and reduction of costs. In this context, we covalently linked proteases from Aspergillus oryzae to polylactic acid an eco-friendly biopolymer. The hydrolytic efficiency of immobilized enzymes was assessed testing their stability to temperature and over time, and checking the hydrolysis of model biomasses (casein and bovine serum albumin). Soybean waste extracts were also used as proof of principle. Full article

Historically, ligase activity by proteases was theoretically derived due to their catalyst nature, and it was experimentally observed as early as around 1900. Initially, the digestive proteases, such as pepsin, chymotrypsin, and trypsin were employed to perform in vitro syntheses of small peptides. Protease-catalyzed ligation is more efficient than peptide bond hydrolysis in organic solvents, representing control of the thermodynamic equilibrium. Peptide esters readily form acyl intermediates with serine and cysteine proteases, followed by peptide bond synthesis at the N-terminus of another residue. This type of reaction is under kinetic control, favoring aminolysis over hydrolysis. Although only a few natural peptide ligases are known, such as ubiquitin ligases, sortases, and legumains, the principle of proteases as general catalysts could be adapted to engineer some proteases accordingly. In particular, the serine proteases subtilisin and trypsin were converted to efficient ligases, which are known as subtiligase and trypsiligase. Together with sortases and legumains, they turned out to be very useful in linking peptides and proteins with a great variety of molecules, including biomarkers, sugars or building blocks with non-natural amino acids. Thus, these engineered enzymes are a promising branch for academic research and for pharmaceutical progress. Full article

The catalytic role of near attack conformations (NACs), molecular states that lie on the pathway between the ground state (GS) and transition state (TS) of a chemical reaction, is not understood completely. Using a computational approach that combines Bürgi–Dunitz theory with all-atom molecular dynamics simulations, the role of NACs in catalyzing the first stages of HIV-1 protease peptide hydrolysis was previously investigated using a substrate that represents the recognized SP1-NC cleavage site of the HIV-1 Gag polyprotein. NACs were found to confer no catalytic effect over the uncatalyzed reaction there ( $\mathsf{\Delta }\mathsf{\Delta }{G}_N^{‡}\sim$ 0 kcal/mol). Here, using the same approach, the role of NACs across multiple substrates that each represent a further recognized cleavage site is investigated. Overall rate enhancement varies by $|\mathsf{\Delta }\mathsf{\Delta }{G}^{‡}|\sim$ 12–15 kcal/mol across this set, and although NACs contribute a small and approximately constant barrier to the uncatalyzed reaction (< $\mathsf{\Delta }$ G ${}_N^{‡u}$ > = 4.3 ± 0.3 kcal/mol), they are found to contribute little significant catalytic effect ( $|\mathsf{\Delta }\mathsf{\Delta }{G}_N^{‡}|\sim$ 0–2 kcal/mol). Furthermore, no correlation is exhibited between NAC contributions and the overall energy barrier ( $R^2$ = 0.01). However, these small differences in catalyzed NAC contributions enable rates to match those required for the kinetic order of processing. Therefore, NACs may offer an alternative and subtle mode compared to non-NAC contributions for fine-tuning reaction rates during complex evolutionary sequence selection processes—in this case across cleavable polyproteins whose constituents exhibit multiple functions during the virus life-cycle. Full article

Bioproduction of vinylphenol derivatives, such as 4-vinylguaiacol (4-VG) and 4-vinylphenol (4-VP), from 4-hydroxycinnamic acids, such as ferulic acid (FA) and p-coumaric acid (pCA), employing whole cells expressing phenolic acid decarboxylases (PAD) as a biocatalyst has attracted much attention in recent years. However, the accumulation of 4-VG or 4-VP in the cell may cause high cytotoxicity to Escherichia coli (E. coli) and consequently cell death during the process. In this study, we firstly report the functional display of a phenolic acid decarboxylase (BLPAD) from Bacillus licheniformis using a GDSL autotransporter from Pseudomonas putida on the cell surface of E. coli. Expression and localization of BLPAD on E. coli were verified by SDS-PAGE and protease accessibility. The PelB signal peptide is more effective in guiding the translocation of BLPAD on the cell surface than the native signal peptide of GDSL, and the cell surface displaying BLPAD activity reached 19.72 U/OD₆₀₀. The cell surface displaying BLPAD showed good reusability and retained 63% of residual activity after 7 cycles of repeated use. In contrast, the residual activity of the intracellular expressing cells was approximately 11% after 3 cycles of reuse. The molar bioconversion yields of 72.6% and 80.4% were achieved at the concentration of 300 mM of FA and pCA in a biphasic toluene/Na₂HPO₄–citric acid buffer system, respectively. Its good reusability and efficient catalysis suggested that the cell surface displaying BLPAD can be used as a whole-cell biocatalyst for efficient production of 4-VG and 4-VP. Full article

Unconstrained amides that undergo fast hydrolysis under mild conditions are valuable sources of information about how amide bonds may be activated in enzymatic transformations. We report a compound possessing an unconstrained amide bond surrounded by an amino and a carboxyl group, each mounted in close proximity on a bicyclic scaffold. Fast amide hydrolysis of this model compound was found to depend on the presence of both the amino and carboxyl functions, and to involve a proton transfer in the rate-limiting step. Possible mechanisms for the hydrolytic cleavage and their relevance to peptide bond cleavage catalyzed by natural enzymes are discussed. Experimental observations suggest that the most probable mechanisms of the model compound hydrolysis might include a twisted amide intermediate and a rate-determining proton transfer. Full article