Enzyme Catalysis Research Articles

A Lysosome-Targeting hNEU1 Inhibitor Treats Myocardial Infarction: A Potential Therapeutic Breakthrough.

The overexpression of NEU1 has recently been certified as being associated with myocardial infarction. However, the pursuit of an efficacious human NEU1 (hNEU1) inhibitor remains challenging, and viral NEU1 (viNEU1) inhibitor drugs are significantly weaker in terms of hNEU1 inhibition. Recognizing that hNEU1 is located within the lysosome, we designed a series of lysosome-targeting compounds, derived from oseltamivir, aimed at hNEU1 inhibition. Among these compounds, OsMo exhibits the most potent activity. Our findings reveal that OsMo accumulates within lysosomes and releases its pharmacophore via enzymatic catalysis. OsMo enhances hNEU1 inhibition by accumulating pharmacophores at the target site. OsMo exhibits improved regulation of abnormal autophagy during myocardial injury, demonstrating superior efficacy in treating myocardial infarction in vivo. Furthermore, OsMo exhibits acceptable pharmacokinetic parameters. Importantly, the development of molecules with lysosome-targeting abilities represents a promising avenue for addressing myocardial injuries linked to hNEU1 overexpression.

Metabolic rewiring during bone development underlies tRNA m7G-associated primordial dwarfism.

Translation of mRNA to protein is tightly regulated by transfer RNAs (tRNAs), which are subject to various chemical modifications that maintain structure, stability, and function. Deficiency of tRNA N7-methylguanosine (m7G) modification in patients causes a type of primordial dwarfism, but the underlying mechanism remains unknown. Here we report that the loss of m7G rewires cellular metabolism, leading to the pathogenesis of primordial dwarfism. Conditional deletion of the catalytic enzyme Mettl1 or missense mutation of the scaffold protein Wdr4 severely impaired endochondral bone formation and bone mass accrual. Mechanistically, Mettl1 knockout decreased abundance of m7G-modified tRNAs and inhibited translation of mRNAs relating to cytoskeleton and Rho GTPase signaling. Meanwhile, Mettl1 knockout enhanced cellular energy metabolism despite incompetent proliferation and osteogenic commitment. Further exploration revealed that impairment of Rho GTPase signaling upregulated the level of branched-chain amino acid transaminase 1 (BCAT1) that rewired cell metabolism and restricted intracellular α-ketoglutarate (αKG). Supplementation of αKG ameliorated the skeletal defect of Mettl1-deficient mice. In addition to the selective translation of metabolism-related mRNAs, we further revealed that Mettl1 knockout globally regulated translation via integrated stress response (ISR) and mammalian target of rapamycin complex 1 (mTORC1) signaling. Restoring translation by targeting either ISR or mTORC1 aggravated bone defects of Mettl1-deficient mice. Overall, our study unveils a critical role of m7G tRNA modification in bone development by regulation of cellular metabolism and indicates suspension of translation initiation as a quality control mechanism in response to tRNA dysregulation.

Utilizing Molecular Dynamics Simulations, Machine Learning, Cryo-EM, and NMR Spectroscopy to Predict and Validate Protein Dynamics.

Protein dynamics play a crucial role in biological function, encompassing motions ranging from atomic vibrations to large-scale conformational changes. Recent advancements in experimental techniques, computational methods, and artificial intelligence have revolutionized our understanding of protein dynamics. Nuclear magnetic resonance spectroscopy provides atomic-resolution insights, while molecular dynamics simulations offer detailed trajectories of protein motions. Computational methods applied to X-ray crystallography and cryo-electron microscopy (cryo-EM) have enabled the exploration of protein dynamics, capturing conformational ensembles that were previously unattainable. The integration of machine learning, exemplified by AlphaFold2, has accelerated structure prediction and dynamics analysis. These approaches have revealed the importance of protein dynamics in allosteric regulation, enzyme catalysis, and intrinsically disordered proteins. The shift towards ensemble representations of protein structures and the application of single-molecule techniques have further enhanced our ability to capture the dynamic nature of proteins. Understanding protein dynamics is essential for elucidating biological mechanisms, designing drugs, and developing novel biocatalysts, marking a significant paradigm shift in structural biology and drug discovery.

Nondissociative Activated Dihydrogen Binding on CeO2 Revealed by High-Pressure Operando Solid-State NMR Spectroscopy.

Dihydrogen complexes, which retain the H-H bond, have been extensively studied in molecular science and found to be prevalent in homogeneous and enzymatic catalysis. However, their counterparts in heterogeneous catalysis, specifically nondissociative chemisorbed dihydrogen binding on the catalyst surface, are rarely reported experimentally. This scarcity is due to the complexity of typical material surfaces and the lack of effective characterization techniques to prove and distinguish various dihydrogen binding modes. Herein, using high-pressure operando solid-state NMR technology, we report the first unambiguous experimental observation of activated dihydrogen binding on a reduced ceria catalyst through interactions with surface oxygen vacancies. By employing versatile NMR structural and dynamical analysis methods, we establish a proportional relationship between the degree of ceria surface reduction and dihydrogen binding, as evidenced by NMR observations of H-D through-bond coupling (JHD), T1 relaxation, and proton isotropic chemical shifts. In situ NMR analysis further reveals the participation of bound dihydrogen species in a room-temperature ethylene hydrogenation reaction. The remarkable similarities between surface-activated dihydrogen in heterogeneous catalysis and dihydrogen model molecular complexes can provide valuable insights into the hydrogenation mechanism for many other solid catalysts, potentially enhancing hydrogen utilization.

Enhanced Selectivity in Microdroplet-Mediated Enzyme Catalysis.

Natural enzymes with enhanced catalytic activity and selectivity have long been studied by tuning the microenvironment around the active site, but how to modulate the active-site electric field in a simple fashion remains challenging. Here, we demonstrate that microdroplets as a simple yet versatile reactor can enhance the electric field at the active site of an enzyme. By using horseradish peroxidase as a model, improved selectivity in microdroplet-mediated enzyme catalysis can be obtained. Quantum mechanical/molecular dynamics calculations and vibrational Stark spectroscopy reveal that the electric field at the microdroplet interface can influence the electrostatic preorganization and orientation of the enzyme to enhance its internal electric field. As a result, the free energies of the substrate and heme can be tuned by the internal electric field, thereby changing its catalytic reaction pathway for a classical substrate, 3,3',5,5'-tetramethylbenzidine, and enabling selective C-N additions for specific substrates. This finding provides a green, simple, and effective way to modulate enzyme-catalyzed reactions and holds promise for a broad spectrum of biosensing and biosynthesis applications.

Computational insights into chemoselectivity of Trans-4-Hydroxy-L-Proline dehydratase HypD

The dehydration of trans-4-hydroxy-L-proline (t4L-HP) yielding 1-pyrroline-5-carboxylate in certain gut bacteria requires the glycyl radical enzyme HypD, while isomerisation of trans-4-hydroxy-D-proline (t4D-HP) generating 2-amino-4-ketopentanoate involves a homologous enzyme HplG. Although the former enzyme exhibits high chemoselectivity when catalysing the dehydration and is regarded as a promising target for treating bacteria-caused gut diseases, its catalytic mechanism remains elusive. Herein, we deeply investigated the two reaction pathways by carrying out theoretical studies at the QM/MM level. Our calculations show that, in HypD, a catalytic Cys434 radical facilitates the Cδ–H activation in an initially deprotonated zwitterionic t4L-HP, which is followed by a heterolytic cleavage of t4L-HP’s Cγ–O bond with a water formed being assisted by the conserved Asp279. In the isomerisation pathway, the Cys434 radical would abstract the hydrogen atom from Cγ of zwitterionic t4L-HP, and then Asp279 cleaves the Cδ–N bond by abstracting a proton from the hydroxyl group of t4L-HP. The two reaction paths are calculated to have barriers of 18.0 and 22.8 kcal/mol, respectively, which well explain HypD’s chemoselectivity. In the mechanisms suggested by our study, Asp279 plays a role as “proton shuttle” and regulates the protonation state of t4L-HP in the selective hydrogen atom abstraction invoked by the cysteine radical. Our findings provide an insight of universal significance into radical-based enzymatic catalysis and expand the understanding of how nature wisely modulates catalytic reactivity and selectivity by switching the substrate’s protonation state.

Aggregation-induced emission-based competitive immunoassays for “signal-on” detection of proteins with multifunctional metal–organic frameworks as signal tags

An aggregation-induced emission (AIE)-based strategy was proposed for fluorescence immunoassays of protein biomarkers using Cu-based metal–organic frameworks (Cu-MOFs) to load recombinant targets and enzymes for dual signal amplification. The immunosensing platform was built based on the sequestration and consumption of the substrates of pyrophosphate (PPi) ions by Cu-MOFs and enzymatic catalysis. The negatively charged PPi could trigger the aggregation of positively charged tetraphenylethene (TPE)-substituted pyridinium salt nanoparticles (TPE-Py NPs) by electrostatic interactions, lighting up the fluorescence due to the AIE phenomenon. The consumption of PPi by the captured Cu-MOFs through the Cu2+-PPi chelation interaction and ALP–enzymatic hydrolysis depressed the aggregation of TPE-Py NPs. Capture of the tested targets in samples by the antibodies on the plate surface could prevent the attachment of target/ALP-loaded Cu-MOFs due to the competitive immunoreactions. The “signal-on” competitive immunoassay was applied for the detection of procalcitonin (PCT) as the model analyte with a linear range of 0.01–10 pg/mL and a detection limit down to 8 pg/mL. The conceptual integration of AIE with enzymatic and MOFs–based dual signal amplification endowed fluorescence immunoassays with high sensitivity and selectivity. The surface modification of Cu-MOFs with hexahistine (His6)-tagged recombinant proteins through metal coordination interactions should be evaluable for the design of novel biosensors.

In situ enzymatic peptide-based nanomedicine with combined effects for enhanced tumor radio-immunotherapy

Despite the combination of radiotherapy and immunotherapy in clinical practice have shown promising synergistic anti-tumor effect, the immunosuppressive tumor microenvironment (TME) remains to be one of stumbling block impeding the therapeutic efficacy of radio-immunotherapy. In this study, in situ self-assembled peptide-based drug delivery systems with multiple effects were constructed for enhancing tumor radio-immunotherapy. The two peptide-drug conjugates could co-assembled into peptide-based nanomedicine on the cancer cell membrane under enzyme catalysis and continuously release small molecular active drugs in the cytoplasm, which combined action lead YAP phosphorylation and nuclear transfer failure. By simutaneously suppressing glycolysis and reactivating the dysfunctional Hippo pathway, the peptide-based nanomedicine also remitted the immunosuppressive TME by reducing lactate levels and the expression of immunosuppressive factors, respectively. As a result, the pro-tumorigenic M2 phenotype of tumor-associated macrophages (TAMs) was decreased and the anti-tumorigenic M1 phenotype of TAMs was increased. Such a dual intervention strategy from cellular metabolic level and gene transcription level not only inhibited tumor cell proliferation but also reversed the immunosuppressive TME, thereby augmenting the combined efficacy of radiotherapy and immune checkpoint blockade (ICB) therapy. The work is anticipated to contribute novel insights into anti-cancer drug targeted delivery and tumor multi-modal synergistic therapy.

Additional feedforward mechanism of Parkin activation via binding of phospho-UBL and RING0 in trans.

Loss-of-function Parkin mutations lead to early-onset of Parkinson's disease. Parkin is an auto-inhibited ubiquitin E3 ligase activated by dual phosphorylation of its ubiquitin-like (Ubl) domain and ubiquitin by the PINK1 kinase. Herein, we demonstrate a competitive binding of the phospho-Ubl and RING2 domains towards the RING0 domain, which regulates Parkin activity. We show that phosphorylated Parkin can complex with native Parkin, leading to the activation of autoinhibited native Parkin in trans. Furthermore, we show that the activator element (ACT) of Parkin is required to maintain the enzyme kinetics, and the removal of ACT slows the enzyme catalysis. We also demonstrate that ACT can activate Parkin in trans but less efficiently than when present in the cis molecule. Furthermore, the crystal structure reveals a donor ubiquitin binding pocket in the linker connecting REP and RING2, which plays a crucial role in Parkin activity.

Additional feedforward mechanism of Parkin activation via binding of phospho-UBL and RING0 in trans

Loss-of-function Parkin mutations lead to early-onset of Parkinson’s disease. Parkin is an auto-inhibited ubiquitin E3 ligase activated by dual phosphorylation of its ubiquitin-like (Ubl) domain and ubiquitin by the PINK1 kinase. Herein, we demonstrate a competitive binding of the phospho-Ubl and RING2 domains towards the RING0 domain, which regulates Parkin activity. We show that phosphorylated Parkin can complex with native Parkin, leading to the activation of autoinhibited native Parkin in trans. Furthermore, we show that the activator element (ACT) of Parkin is required to maintain the enzyme kinetics, and the removal of ACT slows the enzyme catalysis. We also demonstrate that ACT can activate Parkin in trans but less efficiently than when present in the cis molecule. Furthermore, the crystal structure reveals a donor ubiquitin binding pocket in the linker connecting REP and RING2, which plays a crucial role in Parkin activity.

Genomic synergistic efficient carbon fixation and nitrogen removal induced by excessive inorganic carbon in the anammox-centered coupling system

Given the significance of HCO3- for autotrophic anammox bacteria (AnAOB), excessive HCO3- was always provided in anammox-related systems and engineering applications. However, its impact mechanism on anammox process at genome-level remains unknown. This study firstly established an anammox-centered coupling system that entails heterotrophic partial denitrification (PD) and hydrolytic acidification (A-PDHA) fed mainly with inorganic carbon (high HCO3- concentration and low C/N ratio). Metagenomic binning and metatranscriptomics analyses indicated that high HCO3- concentration enhanced expression of natural most efficient phosphoenolpyruvate (PEP) carboxylase within AnAOB, by up to 30.59 folds. This further induced AnAOB to achieve high-speed carbon-fixing reaction through cross-feeding of phosphate and PEP precursors with heterotrophs. Additionally, the enhanced activity of transporters and catalytic enzymes (up to 4949-fold) induced by low C/N ratio enabled heterotrophs to eliminate extracellular accumulated energy precursors mainly derived from carbon fixation products of AnAOB. This maintained high-speed carbon-fixing reaction within AnAOB and supplemented heterotrophs with organics. Moreover, assimilated energy precursors stimulated nitrogen metabolism enzymes, especially NO2- reductase (968.14 times), in heterotrophs. This established an energy-saving PD-A process mediated by interspecies NO shuttle. These variation resulted in efficient nitrogen removal (>95 %) and reduced external organic carbon demand (67 %) in A-PDHA system. This study unveils the great potential of an anammox-centered autotrophic-heterotrophic coupling system for achieving cost-effective nitrogen removal and enhancing carbon fixation under excessive HCO3- doses.

Enzyme Catalysis Today and the Chemistry of the 21st Century

Enzyme Catalysis Today and the Chemistry of the 21st Century

Range-separated hybrid van der Waals density functionals to describe Cu[formula omitted]O[formula omitted]-complexes

Dimeric copper centers are commonly used to activate molecular oxygen in enzymatic and heterogeneous catalysis where Cu2 is embedded in porous structures. Here we evaluate the ability of recently developed range-separated hybrid van der Waals density functionals to describe activated O2 in a crystal with the Cu2O2 unit and in a ▪ complex embedded in a chabzite zeolite. The electronic and geometrical structure of the Cu2O2 unit depends sensitively on the exchange–correlation functional and a sufficiently large amount of short-ranged Fock-exchange is needed to describe the experimentally observed Cu2O2 structures.

Disposal of used oils as a prospective method of production of biodiesel

The article is devoted to the improvement of the production technology of diesel biofuel from waste oils, which are a significant source of environmental pollution. Environmental pollution is one of the biggest environmental problems of our time. Used oils that are not properly disposed of cause serious environmental problems, including water and soil pollution. The use of waste oils for the production of biofuel is a promising technology that allows reducing the amount of pollution and reducing dependence on fossil fuels. Biodiesel from waste oils has significant environmental, economic and sustainable advantages. The main goal of the article is a detailed analysis of modern technologies for the production of diesel biofuel from waste oils and an assessment of their improvements and impact on ecology, economy and society. The article discusses innovative approaches to the processing of used oils, their purification and preparation for further use. An analysis of the main methods of transesterification, hydrogenation, esterification and enzyme catalysis, as well as the latest technologies, such as ultrasonic and microwave intensification of biofuel production, was carried out. New technological solutions for the preliminary preparation of used oils with a high content of free fatty acids using a combination of acid catalysts are proposed, and a technological scheme of the full production cycle is developed. The rational parameters of the equipment for the preliminary preparation of used oils with a high content of free fatty acids have been determined. Recommended conditions include hydrogenation temperature not higher than 80°С, duration of the process not less than 40 minutes; separation of the water-protein part by centrifugation at a rotation frequency of the centrifuge rotor of 3000 rpm for 20 minutes; the esterification reaction temperature is no more than 60°C; molar ratio of alcohol to oil 9:1; acid catalyst concentration within 1-15%; the intensity of mixing in the reactor is 31.42 s-1; the duration of the process is not less than 120 minutes. It was established that it is advisable to use potassium hydroxide for the transesterification reaction. The use of potassium hydroxide is beneficial because the potassium salts formed during the technological process of diesel biofuel production can be used as mineral fertilizers. According to the results of the research, the optimal parameters for the transesterification reaction were chosen: the amount of methanol - 20% by mass. from the weight of the oil, the KOH 1 catalyst is 1.5%, the temperature of the process is 60°C and the duration is 60-70 minutes. Examples of successful implementation of these technologies in various countries of the world, in particular in Europe, the USA and Asia, are presented. Additionally, recommendations are provided for further research and technology development, including the need to improve waste oil purification methods, optimize transesterification processes, and integrate renewable energy sources. The prospects and challenges of the industry of biofuel production from waste oils are considered, in particular the issues of regulatory support, financial incentives and investment attraction.

Mechanism study of Dual-Emission ratiometric fluorescent pH-Sensitive carbon quantum dots and its application on mornitoring enzymatic catalysis

Carbon dots (CQD) have received significant attention as a novel ratiometric fluorescent pH nanoprobe, owing to their favorable optical properties and excellent biocompatibility. Despite their appealing features, the precise mechanism behind the pH-sensitive photoluminescence of CQDs remains to be fully understood. This study endeavors to unravel the mechanism underlying the pH-responsive ratiometric fluorescence in dual-emission CQDs, synthesized through a one-step hydrothermal method using o-phenylenediamine and oxalic acid as precursors. The resultant CQDs exhibit inherent dual-emission at wavelengths of 383 nm and 566 nm, with the ratiometric fluorescence response tailored by the ratio of precursors, providing a robust tool for pH sensing across a range of 2 to 6. Detailed characterizations, including chemical, morphological, and optical analyses, alongside theoretical insights from time-dependent density functional theory (TD-DFT), elucidate the mechanism underlying the pH-dependent luminescence, attributed to the electron cloud transmission between amide and adjacent carboxyl groups on the CQD surface. The superior performance of these CQDs in real-time pH monitoring is demonstrated through their application in glucose oxidase-catalyzed reactions, showcasing their potential as efficient, reliable nanoprobes for biomedical research and diagnostic applications.

Exploring the role of the residues into catalytic cavity of inulosucrase from Leuconostoc citreum CW28

Inulosucrases are enzymes capable of synthesizing inulin polymers using sucrose as the main substrate. The enzymatic activity relies on the catalytic triad within the active site and residues responsible for substrate recognition and orientation, termed carbohydrate-binding subsites. This study investigates the role of specific residues within the catalytic cavity of a truncated version of IslA4 in enzymatic catalysis. Mutants at residues S425, L499, A602, R618, F619, Y676, Y692, and R696 were constructed and characterized. Characterization results, and in silico structural comparison with other fructansucrases, reveal these residues' functional significance in catalysis. Residue S425 belongs to subsite −1; residues R618 and Y692 are part of subsite +1, and residue R696 belongs to subsites +1 and +2. Residues L499 and A602 are support residues; the former favors the formation of the fructosyl-enzyme intermediate, while the latter stabilizes the acid/base catalyst during catalysis. Residues Y676 and F619 may participate in stabilizing residues at −1/+1 subsites. This study represents the first comprehensive exploration of the structural determinants essential for enzymatic function in the inulosucrase of Leuconostoc citreum, and proposes the identity of residues involved in the −1 to +2 subsites.

Fast Reaction of the Prussian Blue Based Nanozyme "Artificial Peroxidase" with the Substrates: Pre-Steady-State Kinetic Approach.

This letter introduces the pre-steady-state kinetic approach, which is traditional for evaluation of elementary constants in molecular (enzyme) catalysis, for nanozymes. Apparently, the most active peroxidase-mimicking nanozyme based on catalytically synthesized Prussian Blue nanoparticles has been chosen. The elementary constants (k1) for the nanozymes' reduction by an electron-donor substrate (being the fastest stage according to steady-state kinetic data) have been determined by means of stopped-flow spectroscopy. These constants have been found to be dependent on both the size of the nanozyme and the reducing substrate redox potential. For the smallest nanozymes (32 nm in diameter), log(k1) linearly decays with an increase of the substrate redox potential (cotangent value ≈125 mV). On the contrary, for the largest nanozymes with a diameter above 150 nm, k1 is almost independent of it. Moreover, for the substrate with the lowest redox potential (K4[Fe(CN)6]), the rate constant under discussion (k1) is almost independent of the nanozymes' size. Perhaps, the rate of the intrananozyme electron transfer causing bleaching becomes comparative or even lower than that of the nanoparticle interaction with the fastest substrate. Anyway, the elementary constant of nanozyme reduction with potassium ferrocyanide (k1) reaches the value of 1 × 1010 M-1 s-1, which is 3-4 orders of magnitude faster than for enzymes peroxidases. The obtained results obviously demonstrate that the pre-steady-state kinetic approach is able to discover novel advantages of nanozymes from both fundamental and practical points of view.

Research progress in biosynthesis of arbutin

Arbutin, a glycosylated compound of hydroquinone, exists in two forms of β-arbutin and α-arbutin based on the configuration of the glycosidic bond. As a safe and stable whitening agent, arbutin is widely used in cosmetics, and it has antioxidant, antimicrobial, anti-inflammatory, and anti-tumor activities. The production of arbutin by plant extraction faces challenges such as long plant growth periods, complex extraction processes, and low yields. The chemical synthesis of arbutin suffers from harsh reaction conditions, poor stereo-selectivity, and low yields. In recent years, biosynthesis emerges as the most popular method to produce arbutin because of the simple and mild reaction conditions, low costs, and environmental friendliness. This review summarizes the research progress in four biosynthetic strategies for arbutin, including plant conversion, enzyme catalysis, whole-cell catalysis, and microbial fermentation. The advantages and limitations of these biosynthetic strategies are discussed, and future research directions are proposed.

Engineering Escherichia coli for D-allulose biosynthesis from glycerol

D-allulose, a naturally occurring monosaccharide, is present in small quantities in nature. It is considered a valuable low-calorie sweetener due to its low absorption in the digestive tract and zero energy for growth. Most of the recent efforts to produce D-allulose have focused on in vitro enzyme catalysis. However, microbial fermentation is emerging as a promising alternative that offers the advantage of combining enzyme manufacturing and product synthesis within a single bioreactor. Here, a novel approach was proposed for the efficient biosynthesis of D-allulose from glycerol using metabolically engineered Escherichia coli. FbaA, Fbp, AlsE, and A6PP were used to construct the D-allulose synthesis pathway. Subsequently, PfkA, PfkB, and Pgi were disrupted to block the entry of the intermediate fructose-6-phosphate (F6P) into the Embden−Meyerhof−Parnas (EMP) and pentose phosphate (PP) pathways. Additionally, GalE and FryA were inactivated to reduce D-allulose consumption by the cells. Finally, a fed-batch fermentation process was implemented to optimize the performance of the cell factory. As a result, the titer of D-allulose reached 7.02 g/L with a maximum yield of 0.287 g/g.

Broadening The Substrate Scope of Aldolases Through Metagenomic Enzyme Discovery.

Bio-processes based on enzymatic catalysis play a major role in the development of green, sustainable processes, and the discovery of new enzymes is key to this approach. In this work, we analysed ten metagenomes and retrieved 48 genes coding for deoxyribose-5-phosphate aldolases (DERAs, EC 4.1.2.4) using a sequence-based approach. These sequences were recombinantly expressed in Escherichia coli and screened for activity towards a range of aldol additions. Among these, one enzyme, DERA-61, proved to be particularly interesting and catalysed the aldol addition of furfural or benzaldehyde with acetone, butanone and cyclobutanone with unprecedented activity. The product of these reactions, aldols, can find applications as building blocks in the synthesis of biologically active compounds. Screening was carried out to identify optimized reaction conditions targeting temperature, pH, and salt concentrations. Lastly, the kinetics and the stereochemistry of the products were investigated, revealing that DERA-61 and other metagenomic DERAs have superior activity and stereoselectivity when they are provided with non-natural substrates, compared to well-known DERAs.