ALDELE: All-Purpose Deep Learning Toolkits for Predicting the Biocatalytic Activities of Enzymes.

Rapidly predicting enzyme properties for catalyzing specific substrates is essential for identifying potential enzymes for industrial transformations. The demand for sustainable production of valuable industry chemicals utilizing biological resources raised a pressing need to speed up biocatalyst screening using machine learning techniques. In this research, we developed an all-purpose deep-learning-based multiple-toolkit (ALDELE) workflow for screening enzyme catalysts. ALDELE incorporates both structural and sequence representations of proteins, alongside representations of ligands by subgraphs and overall physicochemical properties. Comprehensive evaluation demonstrated that ALDELE can predict the catalytic activities of enzymes, and particularly, it identifies residue-based hotspots to guide enzyme engineering and generates substrate heat maps to explore the substrate scope for a given biocatalyst. Moreover, our models notably match empirical data, reinforcing the practicality and reliability of our approach through the alignment with confirmed mutation sites. ALDELE offers a facile and comprehensive solution by integrating different toolkits tailored for different purposes at affordable computational cost and therefore would be valuable to speed up the discovery of new functional enzymes for their exploitation by the industry.

Modular Synthesis of Benzoylpyridines Exploiting a Reductive Arylation Strategy.

Herein we disclose a telescoped flow strategy to access electronically differentiated bisaryl ketones as potentially new and tunable photosensitizers containing both electron-rich benzene systems and electron-deficient pyridyl moieties. Our approach merges a light-driven (365 nm) and catalyst-free reductive arylation between aromatic aldehydes and cyanopyridines with a subsequent oxidation process. The addition of electron-donating and withdrawing substituents on the scaffold allowed effective modification of the absorbance of these compounds in the UV-vis region, while the continuous flow process affords high yields, short residence time, and high throughput.

Origin of the enantioselectivity of alcohol dehydrogenase.

Alcohol dehydrogenases (ADH) are a family of enzymes that catalyse the interconversion between ketones/aldehydes and alcohols in the presence of NADPH cofactor. It is challenging to desymmetrise the substituted cyclopentane-1,3-dione by engineering an ADH, while the reaction mechanism of the metal independent ADH remains elusive. Here we measured the conversion of a model substrate 2-benzyl-2-methylcyclopentane-1,3-dione by LbADH and found it predominately gave the (2R,3R) product. Binding mode analysis of the substrate in LbADH from molecular dynamics simulations disclosed the origin of the enantioselectivity of the enzyme; the opening and closing of the loop 191-205 above the substrate are responsible for shaping the binding pocket to orientate the substrate, so as to give different stereoisomer products. Using QM/MM calculations, we elucidated the reaction mechanism of LbADH. Furthermore, we demonstrated the reaction profile corresponding to the production of different stereoisomers, which is in accordance with our experimental observations. This research here will shed a light on the rational engineering of ADH to achieve stereodivergent stereoisomer products.

Nitrilase mediated mild hydrolysis of a carbon-14 nitrile for the radiosynthesis of 4-(7-hydroxycarbamoyl-[1-14 C-heptanoyl]-oxy)-benzoic acid methyl ester, [14 C]-SHP-141: A novel class I/II histone deacetylase (HDAC) inhibitor.

A strategy has been developed for the carbon-14 radiosynthesis of [14 C]-SHP-141, a 4-(7-hydroxycarbamoyl-heptanoyloxy)-benzoic acid methyl ester derivative containing a terminal hydroxamic acid. The synthesis involved four radiochemical transformations. The key step in the radiosynthesis was the conversion of the 7-[14 C]-cyano-heptanoic acid benzyloxyamide [14 C]-4 directly into the carboxylic acid derivative, 7-benzyloxycarbamoyl-[14 C]-heptanoic acid [14 C]-8 using nitrilase-113 biocatalyst. The final step involved deprotection of the benzyloxy group using catalytic hydrogenation to facilitate the release of the hydroxamic acid without cleaving the phenoxy ester. [14 C]-SHP-141 was isolated with a radiochemical purity of 90% and a specific activity of 190 µCi/mg from four radiochemical steps starting from potassium [14 C]-cyanide in a radiochemical yield of 45%.

Discovery and Rational Mutagenesis of Methionine Sulfoxide Reductase Biocatalysts To Expand the Substrate Scope of the Kinetic Resolution of Chiral Sulfoxides.

Methionine sulfoxide reductase A (MsrA) enzymes have recently found applications as nonoxidative biocatalysts in the enantioselective kinetic resolution of racemic sulfoxides. This work describes the identification of selective and robust MsrA biocatalysts able to catalyze the enantioselective reduction of a variety of aromatic and aliphatic chiral sulfoxides at 8-64 mM concentration with high yields and excellent ees (up to 99%). Moreover, with the aim to expand the substrate scope of MsrA biocatalysts, a library of mutant enzymes has been designed via rational mutagenesis utilizing in silico docking, molecular dynamics, and structural nuclear magnetic resonance (NMR) studies. The mutant enzyme MsrA33 was found to catalyze the kinetic resolution of bulky sulfoxide substrates bearing non-methyl substituents on the sulfur atom with ees up to 99%, overcoming a significant limitation of the currently available MsrA biocatalysts.

Convergent Biocatalytic Mediated Synthesis of siRNA.

New technologies are required to combat the challenges faced with manufacturing commercial quantities of oligonucleotide drug substances which are required for treating large patient populations. Herein we report a convergent biocatalytic synthesis strategy for an Alnylam model siRNA. The siRNA chemical structure includes several of the unnatural modifications and conjugations typical of siRNA drug substances. Using Almac's 3-2-3-2 hybrid RNA ligase enzyme strategy that sequentially ligates short oligonucleotide fragments (blockmers), the target siRNA was produced to high purity at 1 mM concentration. Additional strategies were investigated including the use of polynucleotide kinase phosphorylation and the use of crude blockmer starting materials without chromatographic purification. These findings highlight a path toward a convergent synthesis of siRNAs for large-scale manufacture marrying both enzymatic liquid and classical solid-phase synthesis.

Continuous Flow Epoxidation of Alkenes Using a Homogeneous Manganese Catalyst with Peracetic Acid.

Epoxidation of alkenes is a valuable transformation in the synthesis of fine chemicals. Described herein are the design and development of a continuous flow process for carrying out the epoxidation of alkenes with a homogeneous manganese catalyst at metal loadings as low as 0.05 mol%. In this process, peracetic acid is generated in situ and telescoped directly into the epoxidation reaction, thus reducing the risks associated with its handling and storage, which often limit its use at scale. This flow process lessens the safety hazards associated with both the exothermicity of this epoxidation reaction and the use of the highly reactive peracetic acid. Controlling the speciation of manganese/2-picolinic acid mixtures by varying the ligand:manganese ratio was key to the success of the reaction. This continuous flow process offers an inexpensive, sustainable, and scalable route to epoxides.

Discovery of New Carbonyl Reductases Using Functional Metagenomics and Applications in Biocatalysis.

Enzyme discovery for use in the manufacture of chemicals, requiring high stereoselectivities, continues to be an important avenue of research. Here, a sequence directed metagenomics approach is described to identify short chain carbonyl reductases. PCR from a metagenomic template generated 37 enzymes, with an average 25% sequence identity, twelve of which showed interesting activities in initial screens. Six of the most productive enzymes were then tested against a panel of 21 substrates, including bulkier substrates that have been noted as challenging in biocatalytic reductions. Two enzymes were selected for further studies with the Wieland Miescher ketone. Notably, enzyme SDR-17, when co-expressed with a co-factor recycling system produced the anti-(4aR,5S) isomer in excellent isolated yields of 89% and 99% e.e. These results demonstrate the viability of a sequence directed metagenomics approach for the identification of multiple homologous sequences with low similarity, that can yield highly stereoselective enzymes with applicability in industrial biocatalysis.

Recent Trends in Enzyme Immobilization-Concepts for Expanding the Biocatalysis Toolbox.

Enzymes have been exploited by humans for thousands of years in brewing and baking, but it is only recently that biocatalysis has become a mainstream technology for synthesis. Today, enzymes are used extensively in the manufacturing of pharmaceuticals, food, fine chemicals, flavors, fragrances and other products. Enzyme immobilization technology has also developed in parallel as a means of increasing enzyme performance and reducing process costs. The aim of this review is to present and discuss some of the more recent promising technical developments in enzyme immobilization, including the supports used, methods of fabrication, and their application in synthesis. The review highlights new support technologies such as the use of well-established polysaccharides in novel ways, the use of magnetic particles, DNA, renewable materials and hybrid organic-inorganic supports. The review also addresses how immobilization is being integrated into developing biocatalytic technology, for example in flow biocatalysis, the use of 3D printing and multi-enzymatic cascade reactions.

Interrupted Curtius Rearrangements of Quaternary Proline Derivatives: A Flow Route to Acyclic Ketones and Unsaturated Pyrrolidines.

Conversion of N-Boc-protected quaternary proline derivatives under thermal Curtius rearrangement conditions was found to afford a series of ring-opened ketone and unsaturated pyrrolidine products instead of the expected carbamate species. The nature of the substituent on the quaternary carbon thereby governs the product outcome due to the stability of a postulated N-acyliminium species. A continuous flow process with in-line scavenging was furthermore developed to streamline this transformation and safely create products on a gram scale.

Coupling biocatalysis with high-energy flow reactions for the synthesis of carbamates and ß-amino acid derivatives.

A continuous flow process is presented that couples a Curtius rearrangement step with a biocatalytic impurity tagging strategy to produce a series of valuable Cbz-carbamate products. Immobilized CALB was exploited as a robust hydrolase to transform residual benzyl alcohol into easily separable benzyl butyrate. The resulting telescoped flow process was effectively applied across a series of acid substrates rendering the desired carbamate structures in high yield and purity. The derivatization of these products via complementary flow-based Michael addition reactions furthermore demonstrated the creation of ß-amino acid species. This strategy thus highlights the applicability of this work towards the creation of important chemical building blocks for the pharmaceutical and speciality chemical industries.

Unconventional Biocatalytic Approaches to the Synthesis of Chiral Sulfoxides.

Sulfoxides are a class of organic compounds that find wide application in medicinal and organic chemistry. Several biocatalytic approaches have been developed to synthesise enantioenriched sulfoxides, mainly by exploiting oxidative enzymes. Recently, the use of reductive enzymes such as Msr and Dms has emerged as a new, alternative method to obtain enantiopure sulfoxides from racemic mixtures. In parallel, novel oxidative approaches, employing nonclassical solvents such as ionic liquids (ILs) and deep eutectic solvents (DESs), have been developed as greener and more sustainable biocatalytic synthetic pathways. This minireview aims highlights the recent advances made in the biocatalytic synthesis of enantioenriched sulfoxides by employing such unconventional approaches.

A mild and chemoselective CALB biocatalysed synthesis of sulfoxides exploiting the dual role of AcOEt as solvent and reagent.

A mild, chemoselective and sustainable biocatalysed synthesis of sulfoxides has been developed exploiting CALB and using AcOEt with a dual role of more environmentally friendly reaction solvent and enzyme substrate. A series of sulfoxides, including the drug omeprazole, have been synthesised in high yields and with excellent E-factors.

Regio- and stereoselectivity in the CYP450BM3-catalyzed hydroxylation of complex terpenoids: a QM/MM study.

The site-selective C-H oxidation of terpenoids by P450 attracts great attention because of their wide range of biological activities. However, the binding and catalytic mechanism of P450 for the hydroxylation of complex terpenoid substrates remains elusive, which has limited the rational engineering of P450 as a biocatalyst for terpenoid biosynthesis. Here, we studied the origin of the selectivity and reactivity of P450BM3 in the hydroxylation of terpenoids by combining molecular dynamics simulations and QM/MM calculations, using artemisinin as a model compound. We found that the conformational change of the ß1 sheet at the substrate entrance and the displacement of the ß' helix were critical for reshaping the binding pocket to modulate substrate entrance and positioning the C-H to be activated toward the oxidative species of P450 for the subsequent hydrogen abstraction, the rate-determining step of hydroxylation. There is a distinct linear correlation between activation barriers and reaction coordinates, indicating that reaction coordinates can be used as a facile descriptor for predicting the reactivity of P450BM3. These findings would provide valuable guidance for predicting the selectivity and reactivity of P450BM3 for the selective hydroxylation of non-native terpenoid substrates so as to prioritize the rationally designed enzymes for terpenoid biosynthesis.

Fluorescent molecular logic gates based on photoinduced electron transfer (PET) driven by a combination of atomic and biomolecular inputs.

Molecular AND logic gates 1, 3, 5 and 7, which are designed according to principles of photoinduced electron transfer (PET) switching, respond to co-existing Candida antarctica lipase B and H+ (and Na+).

Transaminases for industrial biocatalysis: novel enzyme discovery.

Transaminases (TAms) are important enzymes for the production of chiral amines for the pharmaceutical and fine chemical industries. Novel TAms for use in these industries have been discovered using a range of approaches, including activity-guided methods and homologous sequence searches from cultured microorganisms to searches using key motifs and metagenomic mining of environmental DNA libraries. This mini-review focuses on the methods used for TAm discovery over the past two decades, analyzing the changing trends in the field and highlighting the advantages and drawbacks of the respective approaches used. This review will also discuss the role of protein engineering in the development of novel TAms and explore possible directions for future TAm discovery for application in industrial biocatalysis. KEY POINTS: • The past two decades of TAm enzyme discovery approaches are explored. • TAm sequences are phylogenetically analyzed and compared to other discovery methods. • Benefits and drawbacks of discovery approaches for novel biocatalysts are discussed. • The role of protein engineering and future discovery directions is highlighted.

Sequential Palladium-Catalyzed Allylic Alkylation/retro-Dieckmann Fragmentation Strategy for the Synthesis of α-Substituted Acrylonitriles.

A straightforward synthesis of α-substituted acrylonitriles is described using 4-cyano-3-oxotetrahydro-thiophene (c-THT) as an acrylonitrile surrogate. This unprecedented two-step sequence featuring a palladium-catalyzed allylic alkylation (Pd-AA) and a retro-Dieckmann fragmentation provides a general entry into diversely substituted 1,4-dienes.

Reaction Mechanism of Histone Demethylation in αKG-dependent Non-Heme Iron Enzymes.

Histone demethylases (KDMs) catalyze histone lysine demethylation, an important epigenetic process that controls gene expression in eukaryotes, and represent important cancer drug targets for cancer treatment. Demethylation of histone is comprised of sequential reaction steps including oxygen activation, decarboxylation, and demethylation. The initial oxygen binding and activation steps have been studied. However, the information on the complete catalytic reaction cycle is limited, which has impeded the structure-based design of inhibitors targeting KDMs. Here we report the mechanism of the complete reaction steps catalyzed by a representative nonheme iron αKG-dependent KDM, PHF8 using QM/MM approaches. The atomic-level understanding on the complete reaction mechanism of PHF8 would shed light on the structure-based design of selective inhibitors targeting KDMs to intervene in cancer epigenetics.

Artificial cysteine-lipases with high activity and altered catalytic mechanism created by laboratory evolution.

Engineering artificial enzymes with high activity and catalytic mechanism different from naturally occurring enzymes is a challenge in protein design. For example, many attempts have been made to obtain active hydrolases by introducing a Ser → Cys exchange at the respective catalytic triads, but this generally induced a breakdown of activity. We now report that this long-standing dogma no longer pertains, provided additional mutations are introduced by directed evolution. By employing Candida antarctica lipase B (CALB) as the model enzyme with the Ser-His-Asp catalytic triad, a highly active cysteine-lipase having a Cys-His-Asp catalytic triad and additional mutations W104V/A281Y/A282Y/V149G can be evolved, showing a 40-fold higher catalytic efficiency than wild-type CALB in the hydrolysis of 4-nitrophenyl benzoate, and tolerating bulky substrates. Crystal structures, kinetics, MD simulations and QM/MM calculations reveal dynamic features and explain all results, including the preference of a two-step mechanism involving the zwitterionic pair Cys105-/His224+ rather than a concerted process.

Mechanism of Phosphatidylglycerol Activation Catalyzed by Prolipoprotein Diacylglyceryl Transferase.

Lipoproteins are essential for bacterial survival. Bacterial lipoprotein biosynthesis is accomplished by sequential modification by three enzymes in the inner membrane, all of which are emerging antimicrobial targets. The X-ray crystal structure of prolipoprotein diacylglyceryl transferase (Lgt) and apolipoprotein N-acyl transferase (Lnt) has been reported. However, the mechanisms of the post-translational modification catalyzed by these enzymes have not been understood. Here, we studied the mechanism of the transacylation reaction catalyzed by Lgt, the first enzyme for lipoprotein modification using molecular docking, molecular dynamics, and quantum mechanics/molecular mechanics (QM/MM) calculations. Our results suggest that Arg143, Arg239, and Glu202 play a critical role in stabilizing the glycerol-1-phosphate head group and activating the glycerol C3-O ester bond of the phosphatidylglycerol (PG) substrate. With PG binding, the opening of the L6-7 loop mediated by the highly conserved Arg236 residue as a gatekeeper is observed, which facilitates the release of the modified lipoprotein product, as well as the entry of another PG substrate. Further QM/MM studies revealed that His103 acts as a catalytic base to abstract a proton from the cysteine residue of the preproliprotein, initiating the diacylglyceryl transfer from PG to preprolipoprotein. This is the first study on the mechanism of lipoprotein modification catalyzed by a post-translocational processing enzyme. The transacylation mechanism of Lgt would shed light on the development of novel antimicrobial therapies targeting the challenging enzymes involved in the post-translocational modification pathway of lipoproteins.