Electrostatic Preorganization in Three Distinct Heterogeneous Proteasome β-Subunits.

The origin of the enzyme's powerful role in accelerating chemical reactions is one of the most critical and still widely discussed questions. It is already accepted that enzymes impose an electrostatic field onto their substrates by adopting complex three-dimensional structures; therefore, the preorganization of electric fields inside protein active sites has been proposed as a crucial contributor to catalytic mechanisms and rate constant enhancement. In this work, we focus on three catalytically active β-subunits of 20S proteasomes with low sequence identity (∼30%) whose active sites, although situated in an electrostatically miscellaneous environment, catalyze the same chemical reaction with similar catalytic efficiency.

Discovery of ICOS-Targeted Small Molecules Using Affinity Selection Mass Spectrometry Screening.

Inducible T cell co-stimulator (ICOS) is a positive immune checkpoint receptor expressed on the surface of activated T cells, which could promote cell function after being stimulated with ICOS ligand (ICOS-L). Although clinical benefits have been reported in the ICOS modulation-based treatment for cancer and autoimmune disease, current modulators are restricted in biologics, whereas ICOS-targeted small molecules are lacking. To fill this gap, we performed an affinity selection mass spectrometry (ASMS) screening for ICOS binding using a library of 15,600 molecules.

Discovery of ICOS-targeted small molecules using affinity selection mass spectrometry screening.

Inducible T cell co-stimulator (ICOS) is a positive immune checkpoint receptor expressed on the surface of activated T cells, which could promote cell function after being stimulated with ICOS ligand (ICOS-L). Although clinical benefits have been reported in the ICOS modulation-based treatment for cancer and autoimmune disease, current modulators are restricted in biologics, whereas ICOS-targeted small molecules are lacking. To fill this gap, we performed an affinity selection mass spectrometry (ASMS) screening for ICOS binding using a library of 15,600 molecules.

Computational study of the mechanism of a polyurethane esterase A (PueA) from .

The effective management of plastic waste has become a global imperative, given our reliance on a linear model in which plastics are manufactured, used once, and then discarded. This has led to the pervasive accumulation of plastic debris in landfills and environmental contamination. Recognizing this issue, numerous initiatives are underway to address the environmental repercussions associated with plastic disposal.

Secondary Amine Catalysis in Enzyme Design: Broadening Protein Template Diversity through Genetic Code Expansion.

Secondary amines, due to their reactivity, can transform protein templates into catalytically active entities, accelerating the development of artificial enzymes. However, existing methods, predominantly reliant on modified ligands or N-terminal prolines, impose significant limitations on template selection. In this study, genetic code expansion was used to break this boundary, enabling secondary amines to be incorporated into alternative proteins and positions of choice.

Electrostatics as a Guiding Principle in Understanding and Designing Enzymes.

Enzyme design faces challenges related to the implementation of the basic principles that govern the catalytic activity in natural enzymes. In this work, we revisit basic electrostatic concepts that have been shown to explain the origin of enzymatic efficiency like preorganization and reorganization. Using magnitudes such as the electrostatic potential and the electric field generated by the protein, we explain how these concepts work in different enzymes and how they can be used to rationalize the consequences of point mutations.

A Single Amino Acid Able to Promote High-Temperature Ring-Opening Polymerization by Dual Activation.

Amino acids are indispensable compounds in the body, performing several biological processes that enable proper functioning. In this work, it is demonstrated that a single amino acid, taurine, is also able to promote the ring-opening polymerization (ROP) of several cyclic monomers under industrially relevant conditions. It is shown that the unique zwitterionic structure of taurine, where the negatively charged sulfonic acid group and the protonated amine group are separated by two methylene groups, not only provides high thermal stability but also leads to a dual activation mechanism, which is corroborated by quantum mechanical calculations.

Peptidyl nitroalkene inhibitors of main protease rationalized by computational and crystallographic investigations as antivirals against SARS-CoV-2.

The coronavirus disease 2019 (COVID-19) pandemic continues to represent a global public health issue. The viral main protease (M) represents one of the most attractive targets for the development of antiviral drugs. Herein we report peptidyl nitroalkenes exhibiting enzyme inhibitory activity against M (K: 1-10 μM) good anti-SARS-CoV-2 infection activity in the low micromolar range (EC: 1-12 μM) without significant toxicity.

Impact of the Warhead of Dipeptidyl Keto Michael Acceptors on the Inhibition Mechanism of Cysteine Protease Cathepsin L.

Cathepsin L (CatL) is a lysosomal cysteine protease whose activity has been related to several human pathologies. However, although preclinical trials using CatL inhibitors were promising, clinical trials have been unsuccessful up to now. We are presenting a study of two designed dipeptidyl keto Michael acceptor potential inhibitors of CatL with either a keto vinyl ester or a keto vinyl sulfone (KVS) warhead.

Mechanistic studies of a lipase unveil effect of pH on hydrolysis products of small PET modules.

Biocatalysis is a key technology enabling plastic recycling. However, despite advances done in the development of plastic-degrading enzymes, the molecular mechanisms that govern their catalytic performance are poorly understood, hampering the engineering of more efficient enzyme-based technologies. In this work, we study the hydrolysis of PET-derived diesters and PET trimers catalyzed by the highly promiscuous lipase B from Candida antarctica (CALB) through QM/MM molecular dynamics simulations supported by experimental Michaelis-Menten kinetics.

Unlocking a (Pseudo)-Mechanically Interlocked Molecule with a Coronene "Shoehorn".

Mechanically interlocked molecules (MIMs) have gained increasing interest during the last decades, not only because of their aesthetic appeal, but also because their unique properties have allowed them to find applications in nanotechnology, catalysis, chemosensing and biomedicine. Herein we describe how a pyrene molecule with four octynyl substituents can be easily encapsulated within the cavity of a tetragold(I) rectangle-like metallobox, by template formation of the metallo-assembly in the presence of the guest. The resulting assembly behaves as a mechanically interlocked molecule (MIM), in which the four long limbs of the guest protrude from the entrances of the metallobox, thus locking the guest inside the cavity of the metallobox.

Engineering a Highly Regioselective Fungal Peroxygenase for the Synthesis of Hydroxy Fatty Acids.

The hydroxylation of fatty acids is an appealing reaction in synthetic chemistry, although the lack of selective catalysts hampers its industrial implementation. In this study, we have engineered a highly regioselective fungal peroxygenase for the ω-1 hydroxylation of fatty acids with quenched stepwise over-oxidation. One single mutation near the Phe catalytic tripod narrowed the heme cavity, promoting a dramatic shift toward subterminal hydroxylation with a drop in the over-oxidation activity.

Combined Theoretical and Experimental Study to Unravel the Differences in Promiscuous Amidase Activity of Two Nonhomologous Enzymes.

Convergent evolution has resulted in nonhomologous enzymes that contain similar active sites that catalyze the same primary and secondary reactions. Comparing how these enzymes achieve their reaction promiscuity can yield valuable insights to develop functions from the optimization of latent activities. In this work, we have focused on the promiscuous amidase activity in the esterase from (Bs2) and compared with the same activity in the promiscuous lipase B from (CALB).

Computational design of an amidase by combining the best electrostatic features of two promiscuous hydrolases.

While there has been emerging interest in designing new enzymes to solve practical challenges, computer-based options to redesign catalytically active proteins are rather limited. Here, a rational QM/MM molecular dynamics strategy based on combining the best electrostatic properties of enzymes with activity in a common reaction is presented. The computational protocol has been applied to the re-design of the protein scaffold of an existing promiscuous esterase from Bs2 to enhance its secondary amidase activity.

Covalent Inhibition of the 20S Proteasome with Homobelactosin C Inquired by QM/MM Studies.

20S proteasome is a main player in the protein degradation pathway in the cytosol, thus intervening in multiple pivotal cellular processes. Over the years the proteasome has emerged as a crucial target for the treatment of many diseases such as neurodegenerative diseases, cancer, autoimmune diseases, developmental disorders, cystic fibrosis, diabetes, cardiac diseases, atherosclerosis, and aging. In this work, the mechanism of proteasome covalent inhibition with bisbenzyl-protected homobelactosin C (hBelC) was explored using quantum mechanics/molecular mechanics (QM/MM) methods.

Impact of Warhead Modulations on the Covalent Inhibition of SARS-CoV-2 M Explored by QM/MM Simulations.

The COVID-19 pandemic, caused by the severe acute respiratory syndrome coronavirus-2, SARS-CoV-2, shows the need for effective antiviral treatments. Here, we present a simulation study of the inhibition of the SARS-CoV-2 main protease (M), a cysteine hydrolase essential for the life cycle of the virus. The free energy landscape for the mechanism of the inhibition process is explored by QM/MM umbrella sampling and free energy perturbation simulations at the M06-2X/MM level of theory for two proposed peptidyl covalent inhibitors that share the same recognition motif but feature distinct cysteine-targeting warheads.

The role of streptavidin and its variants in catalysis by biotinylated secondary amines.

Here, we combine the use of host screening, protein crystallography and QM/MM molecular dynamics simulations to investigate how the protein structure affects iminium catalysis by biotinylated secondary amines in a model 1,4 conjugate addition reaction. Monomeric streptavidin (M-Sav) lacks a quaternary structure and the solvent-exposed reaction site resulted in poor product conversion in the model reaction with low enantio- and regioselectivities. These parameters were much improved when the tetrameric host T-Sav was used; indeed, residues at the symmetrical subunit interface were proven to be critical for catalysis through a mutagenesis study.

Discovery of SARS-CoV-2 M peptide inhibitors from modelling substrate and ligand binding.

The main protease (M) of SARS-CoV-2 is central to viral maturation and is a promising drug target, but little is known about structural aspects of how it binds to its 11 natural cleavage sites. We used biophysical and crystallographic data and an array of biomolecular simulation techniques, including automated docking, molecular dynamics (MD) and interactive MD in virtual reality, QM/MM, and linear-scaling DFT, to investigate the molecular features underlying recognition of the natural M substrates. We extensively analysed the subsite interactions of modelled 11-residue cleavage site peptides, crystallographic ligands, and docked COVID Moonshot-designed covalent inhibitors.

Computational Studies Suggest Promiscuous Lipase B as an Environmentally Friendly Alternative for the Production of Epoxides.

Environmentally friendly processes are nowadays a trending topic to get highly desired chemical compounds and, in this sense, the use of enzyme-catalyzed routes is becoming a promising alternative to traditional synthetic methods. In the present paper, a hybrid quantum mechanics/molecular mechanics (QM/MM) computational study on the epoxidation of alkenes catalyzed by the Ser105Ala variant of the promiscuous lipase B (CALB) is presented in an attempt to search for alternative paths to get useful intermediates in industries. The catalyzed reaction, described at the atomistic level with a model of the full solvated in a box of water molecules, is compared with the alternative epoxidation of alkenes by peroxy acids in chloroform.

Mechanism of inhibition of SARS-CoV-2 M by peptidyl Michael acceptor explained by QM/MM simulations and design of new derivatives with tunable chemical reactivity.

The SARS-CoV-2 main protease (M) is essential for replication of the virus responsible for the COVID-19 pandemic, and one of the main targets for drug design. Here, we simulate the inhibition process of SARS-CoV-2 M with a known Michael acceptor (peptidyl) inhibitor, . The free energy landscape for the mechanism of the formation of the covalent enzyme-inhibitor product is computed with QM/MM molecular dynamics methods.

Revealing the molecular mechanisms of proteolysis of SARS-CoV-2 M by QM/MM computational methods.

SARS-CoV-2 M is one of the enzymes essential for the replication process of the virus responsible for the COVID-19 pandemic. This work is focused on exploring its proteolysis reaction by means of QM/MM methods. The resulting free energy landscape of the process provides valuable information on the species appearing along the reaction path and suggests that the mechanism of action of this enzyme, taking place in four steps, slightly differs from that of other cysteine proteases.

Glycoside hydrolase stabilization of transition state charge: new directions for inhibitor design.

Carbasugars are structural mimics of naturally occurring carbohydrates that can interact with and inhibit enzymes involved in carbohydrate processing. In particular, carbasugars have attracted attention as inhibitors of glycoside hydrolases (GHs) and as therapeutic leads in several disease areas. However, it is unclear how the carbasugars are recognized and processed by GHs.

Caught in Action: X-ray Structure of Thymidylate Synthase with Noncovalent Intermediate Analog.

Methylation of 2-deoxyuridine-5'-monophosphate (dUMP) at the C5 position by the obligate dimeric thymidylate synthase (TSase) in the sole biosynthetic pathway to thymidine 5'-monophosphate (dTMP) proceeds by forming a covalent ternary complex with dUMP and cosubstrate 5,10-methylenetetrahydrofolate. The crystal structure of an analog of this intermediate gives important mechanistic insights but does not explain the half-of-the-sites activity of the enzyme. Recent experiments showed that the C5 proton and the catalytic Cys are eliminated in a concerted manner from the covalent ternary complex to produce a noncovalent bisubstrate intermediate.

Transfer hydrogenations catalyzed by streptavidin-hosted secondary amine organocatalysts.

Here, the streptavidin-biotin technology was applied to enable organocatalytic transfer hydrogenation. By introducing a biotin-tethered pyrrolidine (1) to the tetrameric streptavidin (T-Sav), the resulting hybrid catalyst was able to mediate hydride transfer from dihydro-benzylnicotinamide (BNAH) to α,β-unsaturated aldehydes. Hydrogenation of cinnamaldehyde and some of its aryl-substituted analogues was found to be nearly quantitative.