Engineering cascade biocatalysis in whole cells for syringic acid bioproduction.

BACKGROUND: Syringic acid (SA) is a high-value natural compound with diverse biological activities and wide applications, commonly found in fruits, vegetables, and herbs. SA is primarily produced through chemical synthesis, nonetheless, these chemical methods have many drawbacks, such as considerable equipment requirements, harsh reaction conditions, expensive catalysts, and numerous by-products. Therefore, in this study, a novel biotransformation route for SA production was designed and developed by using engineered whole cells. RESULTS: An O-methyltransferase from Desulfuromonas acetoxidans (DesAOMT), which preferentially catalyzes a methyl transfer reaction on the meta-hydroxyl group of catechol analogues, was identified. The whole cells expressing DesAOMT can transform gallic acid (GA) into SA when S-adenosyl methionine (SAM) is used as a methyl donor. We constructed a multi-enzyme cascade reaction in Escherichia coli, containing an endogenous shikimate kinase (AroL) and a chorismate lyase (UbiC), along with a p-hydroxybenzoate hydroxylase mutant (PobA**) from Pseudomonas fluorescens, and DesAOMT; SA was biosynthesized from shikimic acid (SHA) by using whole cells catalysis. The metabolic system of chassis cells also affected the efficiency of SA biosynthesis, blocking the chorismate metabolism pathway improved SA production. When the supply of the cofactor NADPH was optimized, the titer of SA reached 133 µM (26.2 mg/L). CONCLUSION: Overall, we designed a multi-enzyme cascade in E. coli for SA biosynthesis by using resting or growing whole cells. This work identified an O-methyltransferase (DesAOMT), which can catalyze the methylation of GA to produce SA. The multi-enzyme cascade containing four enzymes expressed in an engineered E. coli for synthesizing of SA from SHA. The metabolic system of the strain and biotransformation conditions influenced catalytic efficiency. This study provides a new green route for SA biosynthesis.

Interplay of structural preorganization and conformational sampling in UDP-glucuronic acid 4-epimerase catalysis.

Understanding enzyme catalysis as connected to protein motions is a major challenge. Here, based on temperature kinetic studies combined with isotope effect measurements, we obtain energetic description of C-H activation in NAD-dependent UDP-glucuronic acid C4 epimerase. Approach from the ensemble-averaged ground state (GS) to the transition state-like reactive conformation (TSRC) involves, alongside uptake of heat ( Δ H ‡ = 54 kJ mol-1), significant loss in entropy ( - T Δ S ‡ = 20 kJ mol-1; 298 K) and negative activation heat capacity ( Δ C p ‡ = -0.64 kJ mol-1 K-1). Thermodynamic changes suggest the requirement for restricting configurational freedom at the GS to populate the TSRC. Enzyme variants affecting the electrostatic GS preorganization reveal active-site interactions important for precise TSRC sampling and H-transfer. Collectively, our study captures thermodynamic effects associated with TSRC sampling and establishes rigid positioning for C-H activation in an enzyme active site that requires conformational flexibility in fulfillment of its natural epimerase function.

Photocatalytic regeneration of nicotinamide cofactor biomimetics drives biocatalytic reduction by Old Yellow enzymes.

A key approach in developing green chemistry involves converting solar energy into chemical energy of biomolecules through photocatalysis. Photocatalysis can facilitate the regeneration of nicotinamide cofactors during redox processes. Nicotinamide cofactor biomimetics (NCBs) are economical substitutes for natural cofactors. Here, photocatalytic regeneration of NADH and reduced NCBs (NCBsred) using graphitic carbon nitride (g-C3N4) was developed. The process involves g-C3N4 as the photocatalyst, Cp*Rh(bpy)H2O2+ as the electron mediator, and Triethanolamine as the electron donor, facilitating the reduction of NAD+ and various oxidative NCBs (NCBsox) under light irradiation. Notably, the highest reduction yield of 48.32 % was achieved with BANA+, outperforming the natural cofactor NAD+. Electrochemical analysis reveals that the reduction efficiency and capacity of cofactors relies on their redox potentials. Additionally, a coupled photo-enzymatic catalysis system was explored for the reduction of 4-Ketoisophorone by Old Yellow Enzyme XenA. Among all the NCBsox and NAD+, the highest conversion ratio of over 99 % was obtained with BANA+. After recycled for 8 times, g-C3N4 maintained over 93.6 % catalytic efficiency. The photocatalytic cofactor regeneration showcases its outstanding performance with NAD+ as well as NCBsox. This work significantly advances the development of photocatalytic cofactor regeneration for artificial cofactors and its potential application.

The Importance of Spin-Polarized Charge Reorganization in the Catalytic Activity of D-Glucose Oxidase.

The front cover artwork is provided by Prof. Ron Naaman's group at the Weizmann Institute of Science. The image shows that direct electron transfer through GOx is governed by electron spins, which result from the chiral-induced spin selectivity (CISS) effect. Read the full text of the Research Article at 10.1002/cphc.202400033.

Discovery of Potent Glycosidases Enables Quantification of Smoke-Derived Phenolic Glycosides through Enzymatic Hydrolysis.

When grapes are exposed to wildfire smoke, certain smoke-related volatile phenols (VPs) can be absorbed into the fruit, where they can be then converted into volatile-phenol (VP) glycosides through glycosylation. These volatile-phenol glycosides can be particularly problematic from a winemaking standpoint as they can be hydrolyzed, releasing volatile phenols, which can contribute to smoke-related off-flavors. Current methods for quantitating these volatile-phenol glycosides present several challenges, including the requirement of expensive capital equipment, limited accuracy due to the molecular complexity of the glycosides, and the utilization of harsh reagents. To address these challenges, we proposed an enzymatic hydrolysis method enabled by a tailored enzyme cocktail of novel glycosidases discovered through genome mining, and the generated VPs from VP glycosides can be quantitated by gas chromatography-mass spectrometry (GC-MS). The enzyme cocktails displayed high activities and a broad substrate scope when using commercially available VP glycosides as the substrates for testing. When evaluated in an industrially relevant matrix of Cabernet Sauvignon wine and grapes, this enzymatic cocktail consistently achieved a comparable efficacy of acid hydrolysis. The proposed method offers a simple, safe, and affordable option for smoke taint analysis.

Reflections on the Origin of Coded Protein Biosynthesis.

The principle of continuity posits that some central features of primordial biocatalytic mechanisms should still be present in the genetically dependent pathway of protein synthesis, a crucial step in the emergence of life. Key bimolecular reactions of this process are catalyzed by DNA-dependent RNA polymerases, aminoacyl-tRNA synthetases, and ribosomes. Remarkably, none of these biocatalysts contribute chemically active groups to their respective reactions. Instead, structural and functional studies have demonstrated that nucleotidic α-phosphate and ß-d-ribosyl 2' OH and 3' OH groups can help their own catalysis, a process which, consequently, has been called "substrate-assisted". Furthermore, upon binding, the substrates significantly lower the entropy of activation, exclude water from these catalysts' active sites, and are readily positioned for a reaction. This binding mode has been described as an "entropy trap". The combination of this effect with substrate-assisted catalysis results in reactions that are stereochemically and mechanistically simpler than the ones found in most modern enzymes. This observation is consistent with the way in which primordial catalysts could have operated; it may also explain why, thanks to their complementary reactivities, ß-d-ribose and phosphate were naturally selected to be the central components of early coding polymers.

Platform-directed allostery and quaternary structure dynamics of SAMHD1 catalysis.

SAMHD1 regulates cellular nucleotide homeostasis, controlling dNTP levels by catalysing their hydrolysis into 2'-deoxynucleosides and triphosphate. In differentiated CD4+ macrophage and resting T-cells SAMHD1 activity results in the inhibition of HIV-1 infection through a dNTP blockade. In cancer, SAMHD1 desensitizes cells to nucleoside-analogue chemotherapies. Here we employ time-resolved cryogenic-EM imaging and single-particle analysis to visualise assembly, allostery and catalysis by this multi-subunit enzyme. Our observations reveal how dynamic conformational changes in the SAMHD1 quaternary structure drive the catalytic cycle. We capture five states at high-resolution in a live catalytic reaction, revealing how allosteric activators support assembly of a stable SAMHD1 tetrameric core and how catalysis is driven by the opening and closing of active sites through pairwise coupling of active sites and order-disorder transitions in regulatory domains. This direct visualisation of enzyme catalysis dynamics within an allostery-stabilised platform sets a precedent for mechanistic studies into the regulation of multi-subunit enzymes.

Avoiding common pitfalls in designing kinetic protocols for catalytic amyloid studies.

Kinetic characterization of catalytic amyloids arguably presents a most challenging type of kinetic experiment where careful consideration of many factors is required. Here we outline common pitfalls in devising kinetic studies in such systems. Unlike the more specific protocols for various applications described in this volume, this chapter deals with general issues in setting up kinetic experiments that are incredibly important but often go without explicit mention in the specialized literature. The kinetic fundamentals described here can be also be of use to the enzymologists working with more traditional catalysts.

A method for in situ self-assembly of the catalytic peptide in enzymatic compartments of glucan particles.

Drawing inspiration from cellular compartmentalization, enzymatic compartments play a pivotal role in bringing enzymes and substrates into confined environments, offering heightened catalytic efficiency and prolonged enzyme lifespan. Previously, we engineered bioinspired enzymatic compartments, denoted as TPE-Q18H@GPs, achieved through the spatiotemporally controllable self-assembly of the catalytic peptide TPE-Q18H within hollow porous glucan particles (GPs). This design strategy allows substrates and products to freely traverse, while retaining enzymatic aggregations. The confined environment led to the formation of catalytic nanofibers, resulting in enhanced substrate binding affinity and a more than two-fold increase in the second-order kinetic constant (kcat/Km) compared to TPE-Q18H nanofibers in a dispersed system. In this work, we will introduce how to synthesize the above-mentioned enzymatic compartments using salt-responsive catalytic peptides and GPs.

Catalytic amyloids for nucleotide hydrolysis.

The design of small peptides that assemble into catalytically active intermolecular structures has proven to be a successful strategy towards developing minimalistic catalysts that exhibit some of the unique functional features of enzymes. Among these, catalytic amyloids have emerged as a fruitful source to unravel many different activities. These assemblies can potentially have broad applications that range from biotechnology to prebiotic chemistry. Although many peptides that assemble into catalytic amyloids have been developed in recent years, the elucidation of convergent mechanistic aspects of the catalysis and the structure/function relationship is still a challenge. Novel catalytic activities are necessary to better address these issues and expand the current repertoire of applicability. In this chapter, we described a methodology to produce catalytic amyloids that are specifically active towards the hydrolysis of phosphoanhydride bonds of nucleotides. The design of potentially active amyloid-prone peptide sequences is explored using as template the active site of enzymes with nucleotidyltransferase activity. The procedures include an approach for sequence design, in vitro aggregation assays, morphological characterization of the amyloid state and a comprehensive methodology to measure activity in vitro using nucleoside and deoxynucleosides triphosphates as model substrates. The proposed strategy can also be implemented to explore different types of activities for the design of future catalytic amyloids.

Simulating chemical reactions promoted by self-assembled peptides with catalytic properties.

Peptides that self-assemble exhibit distinct three-dimensional structures and attributes, positioning them as promising candidates for biocatalysts. Exploring their catalytic processes enhances our comprehension of the catalytic actions inherent to self-assembling peptides, laying a theoretical foundation for creating novel biocatalysts. The investigation into the intricate reaction mechanisms of these entities is rendered challenging due to the vast variability in peptide sequences, their aggregated formations, supportive elements, structures of active sites, types of catalytic reactions, and the interplay between these variables. This complexity hampers the elucidation of the linkage between sequence, structure, and catalytic efficiency in self-assembling peptide catalysts. This chapter delves into the latest progress in understanding the mechanisms behind peptide self-assembly, serving as a catalyst in hydrolysis and oxidation reactions, and employing computational analyses. It discusses the establishment of models, selection of computational strategies, and analysis of computational procedures, emphasizing the application of modeling techniques in probing the catalytic mechanisms of peptide self-assemblies. It also looks ahead to the potential future trajectories within this research domain. Despite facing numerous obstacles, a thorough investigation into the structural and catalytic mechanisms of peptide self-assemblies, combined with the ongoing advancement in computational simulations and experimental methodologies, is set to offer valuable theoretical insights for the development of new biocatalysts, thereby significantly advancing the biocatalysis field.

Metal co-factors to enhance catalytic activity of short prion-derived peptide sequences.

Development of biomolecular enzyme mimics to efficiently catalyse biochemical reactions are of prime relevance for the bulk scale production of industrially relevant biocatalyst. In this regard, amyloidogenic peptides act as suitable self-assembling scaffolds, providing stable nanostructures with high surface area facilitating biocatalysis. Herein, we rationally design two positional amyloidogenic peptide isomers, "Fmoc-VYYAHH (1)" and "Fmoc-VHHAYY (2)" considering catalytic and metal binding affinity of histidine and tyrosine when placed in periphery vs. inner core of the peptide sequence. With an ultimate objective of designing metalloenzyme mimic, we choose Co2+ and Cu2+ as divalent transition metal cations for peptide complexation to aid in catalysis. After optimizing self-assembly of innate peptides, we investigate metal-peptide binding ratio and co-ordination, finally selecting 1:1 peptide metal complex suitable for biocatalysis. Metallopeptides act as better catalysts than the innate peptides as acyl esterase when tyrosines were present at the periphery. Kinetic parameters for assessing hydrolysis rate were calculated by fitting data into Michaelis-Menten and Lineweaver Burk plots. Catalytic activity is altered depending on the stability of peptide metal complexes. 2-Cu acting as the best biocatalyst with a kcat/KM = 0.08 M/s. The protocols mentioned in this chapter meticulously cover the design, synthesis, self-assembly and enzyme kinetics.

Determining the esterase activity of peptides and peptide assemblies.

Catalytic peptides are gaining attention as alternatives to enzymes, especially in industrial applications. Recent advances in peptide design have improved their catalytic efficiency with approaches such as self-assembly and metal ion complexation. However, the fundamental principles governing peptide catalysis at the sequence level are still being explored. Ester hydrolysis, a well-studied reaction, serves as a widely employed method to evaluate the catalytic potential of peptides. The standard colorimetric reaction involving para-nitrophenyl acetate hydrolysis acts as a benchmark assay, providing a straightforward and efficient screening method for rapidly identifying potential catalysts. However, maintaining standardized conditions is crucial for reproducible results, given that factors such as pH, temperature, and substrate concentration can introduce unwanted variability. This necessity becomes particularly pronounced when working with peptides, which often exhibit slower reaction rates compared to enzymes, making even minor variations significantly influential on the final outcome. In this context, we present a refined protocol for assessing the catalytic activity of peptides and peptide assemblies, addressing critical considerations for reproducibility and accuracy.

Boron catalysis in a designer enzyme.

Enzymes play an increasingly important role in improving the benignity and efficiency of chemical production, yet the diversity of their applications lags heavily behind chemical catalysts as a result of the relatively narrow range of reaction mechanisms of enzymes. The creation of enzymes containing non-biological functionalities facilitates reaction mechanisms outside nature's canon and paves the way towards fully programmable biocatalysis1-3. Here we present a completely genetically encoded boronic-acid-containing designer enzyme with organocatalytic reactivity not achievable with natural or engineered biocatalysts4,5. This boron enzyme catalyses the kinetic resolution of hydroxyketones by oxime formation, in which crucial interactions with the protein scaffold assist in the catalysis. A directed evolution campaign led to a variant with natural-enzyme-like enantioselectivities for several different substrates. The unique activation mode of the boron enzyme was confirmed using X-ray crystallography, high-resolution mass spectrometry (HRMS) and 11B NMR spectroscopy. Our study demonstrates that genetic-code expansion can be used to create evolvable enantioselective enzymes that rely on xenobiotic catalytic moieties such as boronic acids and access reaction mechanisms not reachable through catalytic promiscuity of natural or engineered enzymes.

A New Approach in Lipase-Octyl-Agarose Biocatalysis of 2-Arylpropionic Acid Derivatives.

The use of lipase immobilized on an octyl-agarose support to obtain the optically pure enantiomers of chiral drugs in reactions carried out in organic solvents is a great challenge for chemical and pharmaceutical sciences. Therefore, it is extremely important to develop optimal procedures to achieve a high enantioselectivity of the biocatalysts in the organic medium. Our paper describes a new approach to biocatalysis performed in an organic solvent with the use of CALB-octyl-agarose support including the application of a polypropylene reactor, an appropriate buffer for immobilization (Tris base-pH 9, 100 mM), a drying step, and then the storage of immobilized lipases in a climatic chamber or a refrigerator. An immobilized lipase B from Candida antarctica (CALB) was used in the kinetic resolution of (R,S)-flurbiprofen by enantioselective esterification with methanol, reaching a high enantiomeric excess (eep = 89.6 ± 2.0%). As part of the immobilization optimization, the influence of different buffers was investigated. The effect of the reactor material and the reaction medium on the lipase activity was also studied. Moreover, the stability of the immobilized lipases: lipase from Candida rugosa (CRL) and CALB during storage in various temperature and humidity conditions (climatic chamber and refrigerator) was tested. The application of the immobilized CALB in a polypropylene reactor allowed for receiving over 9-fold higher conversion values compared to the results achieved when conducting the reaction in a glass reactor, as well as approximately 30-fold higher conversion values in comparison with free lipase. The good stability of the CALB-octyl-agarose support was demonstrated. After 7 days of storage in a climatic chamber or refrigerator (with protection from humidity) approximately 60% higher conversion values were obtained compared to the results observed for the immobilized form that had not been stored. The new approach involving the application of the CALB-octyl-agarose support for reactions performed in organic solvents indicates a significant role of the polymer reactor material being used in achieving high catalytic activity.

Amination of naringinase to improve citrus juice debittering using a catalyst immobilized on glyoxyl-agarose.

A naringinase complex was chemically aminated prior to its immobilization on glyoxyl-agarose to develop a robust biocatalyst for juice debittering. The effects of amination on the optimal pH and temperature, thermal stability, and debittering performance were analyzed. Concentration of amino groups on catalysts surface increased in 36 %. Amination reduced the ß-glucosidase activity of naringinase complex; however, did not affect optimal pH and temperature of the enzyme and it favored immobilization, obtaining α-l-rhamnosidase and ß-d-glucosidase activities of 1.7 and 4.2 times the values obtained when the unmodified enzymes were immobilized. Amination favored the stability of the immobilized biocatalyst, retaining 100 % of both activities after 190 h at 30 °C and pH 3, while its non-aminated counterpart retained 80 and 52 % of α-rhamnosidase and ß-glucosidase activities, respectively. The immobilized catalyst showed a better performance in grapefruit juice debittering, obtaining a naringin conversion of 7 times the value obtained with the non-aminated catalyst.

Immobilized Dipeptidase in Manganese Ion-Loaded Polyethylenimine-Induced Calcium Phosphate Nanocrystals for Carnosine Synthesis.

Carnosine is a natural bioactive dipeptide with important physiological functions widely used in food and medicine. Dipeptidase (PepD) from Serratia marcescens can catalyze the reverse hydrolytic reaction of ß-alanine with l-histidine to synthesize carnosine in the presence of Mn2+. However, it remains challenging to practice carnosine biosynthesis due to the low activity and high cost of the enzyme. Therefore, the development of biocatalysts with high activity and stability is of significance for carnosine synthesis. Here, we proposed to chelate Mn2+ to polyethylenimine (PEI) that induced rapid formation of calcium phosphate nanocrystals (CaP), and Mn-PEI@CaP was used for PepD immobilization via electrostatic interaction. Mn-PEI@CaP as the carrier enhanced the stability of the immobilized enzyme. Moreover, Mn2+ loaded in the carrier acted as an in situ activator of the immobilized PepD for facilitating the biocatalytic process of carnosine synthesis. The as-prepared immobilized enzyme (PepD-Mn-PEI@CaP) kept similar activity with free PepD plus Mn2+ (activity recovery, 102.5%), while exhibiting elevated thermal stability and pH tolerance. Moreover, it exhibited about two times faster carnosine synthesis than the free PepD system. PepD-Mn-PEI@CaP retained 86.8% of the original activity after eight cycles of batch catalysis without the addition of free Mn2+ ions during multiple cycles. This work provides a new strategy for the co-immobilization of PepD and Mn2+, which greatly improves the operability of the biocatalysis and demonstrates the potential of the immobilized PepD system for efficient carnosine synthesis.

An In Vitro Hybrid Biocatalytic System Enabled by a Combination of Surface-Displayed, Purified, and Cell-Free Expressed Enzymes.

Enzymatic cascades have become a green and sustainable approach for the synthesis of valuable chemicals and pharmaceuticals. Using sequential enzymes to construct a multienzyme complex is an effective way to enhance the overall performance of biosynthetic routes. Here we report the design of an efficient in vitro hybrid biocatalytic system by assembling three enzymes that can convert styrene to (S)-1-phenyl-1,2-ethanediol. Specifically, we prepared the three enzymes in different ways, which were cell surface-displayed, purified, and cell-free expressed. To assemble them, we fused two orthogonal peptide-protein pairs (i.e., SpyTag/SpyCatcher and SnoopTag/SnoopCatcher) to the three enzymes, allowing their spatial organization by covalent assembly. By doing this, we constructed a multienzyme complex, which could enhance the production of (S)-1-phenyl-1,2-ethanediol by 3 times compared to the free-floating enzyme system without assembly. After optimization of the reaction system, the final product yield reached 234.6 µM with a substrate conversion rate of 46.9% (based on 0.5 mM styrene). Taken together, our strategy integrates the merits of advanced biochemical engineering techniques, including cellular surface display, spatial enzyme organization, and cell-free expression, which offers a new solution for chemical biosynthesis by enzymatic cascade biotransformation. We, therefore, anticipate that our approach will hold great potential for designing and constructing highly efficient systems to synthesize chemicals of agricultural, industrial, and pharmaceutical significance.

Elucidation of the Catalytic Sequence of Dihydroorotate Dehydrogenase B from Lactoccocus lactis: Evidence for Accumulation of a Flavin Bisemiquinone State in Catalysis.

The physiological role of dihydroorotate dehydrogenase (DHOD) enzymes is to catalyze the oxidation of dihydroorotate to orotate in pyrimidine biosynthesis. DHOD enzymes are structurally diverse existing as both soluble and membrane-associated forms. The Family 1 enzymes are soluble and act either as conventional single subunit flavin-dependent dehydrogenases known as Class 1A (DHODA) or as unusual heterodimeric enzymes known as Class 1B (DHODB). DHODBs possess two active sites separated by ∼20 Å, each with a noncovalently bound flavin cofactor. NAD is thought to interact at the FAD containing site, and the pyrimidine substrate is known to bind at the FMN containing site. At the approximate center of the protein is a single Fe2S2 center that is assumed to act as a conduit, facilitating one-electron transfers between the flavins. We present anaerobic transient state analysis of a DHODB enzyme from Lactoccocus lactis. The data presented primarily report the exothermic reaction that reduces orotate to dihydroorotate. The reductive half reaction reveals rapid two-electron reduction that is followed by the accumulation of a four-electron reduced state when NADH is added in excess, suggesting that the initial two electrons acquired reside on the FMN cofactor. Concomitant with the first reduction is the accumulation of a long-wavelength absorption feature consistent with the blue form of a flavin semiquinone. Spectral deconvolution and fitting to a model that includes reversibility for the second electron transfer reveals equilibrium accumulation of a flavin bisemiquinone state that has features of both red and blue semiquinones. Single turnover reactions with limiting NADH and excess orotate reveal that the flavin bisemiquinone accumulates with reduction of the enzyme by NADH and decays with reduction of the pyrimidine substrate, establishing the bisemiquinone as a fractional state of the two-electron reduced intermediate observed.

Myosin-Catalyzed ATP Hydrolysis in the Presence of Disease-Causing Mutations: Mavacamten as a Way to Repair Mechanism.

Hypertrophic cardiomyopathy is one of the most common forms of genetic cardiomyopathy. Mavacamten is a first-in-class myosin modulator that was identified via activity screening on the wild type, and it is FDA-approved for the treatment of obstructive hypertrophic cardiomyopathy (HCM). The drug selectively binds to the cardiac ß-myosin, inhibiting myosin function to decrease cardiac contractility. Though the drug is thought to affect multiple steps of the myosin cross-bridge cycle, its detailed mechanism of action is still under investigation. Individual steps in the overall cross-bridge cycle must be queried to elucidate the full mechanism of action. In this study, we utilize the rare-event method of transition path sampling to generate reactive trajectories to gain insights into the action of the drug on the dynamics and rate of the ATP hydrolysis step for human cardiac ß-myosin. We study three known HCM causative myosin mutations: R453C, P710R, and R712L to observe the effect of the drug on the alterations caused by these mutations in the chemical step. Since the crystal structure of the drug-bound myosin was not available at the time of this work, we created a model of the drug-bound system utilizing a molecular docking approach. We find a significant effect of the drug in one case, where the actual mechanism of the reaction is altered from the wild type by mutation. The drug restores both the rate of hydrolysis to the wildtype level and the mechanism of the reaction. This is a way to check the effect of the drug on untested mutations.