Nature, Published online: 01 June 2022; doi:10.1038/s41586-022-04456-z
Recent progress in computational enzyme design, active site engineering and directed evolution are reviewed, highlighting methodological innovations needed to deliver improved designer biocatalysts.R.B. Leveson-Gower
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[ASAP] 19F Electron-Nuclear Double Resonance Reveals Interaction between Redox-Active Tyrosines across the α/β Interface of E. coli Ribonucleotide Reductase
Discovery of non-squalene triterpenes
Nature, Published online: 01 June 2022; doi:10.1038/s41586-022-04773-3
Chimeric triterpene synthases are identified that catalyse non-squalene-dependent triterpene biosynthesis.Synthesis of Oxygenated Sesquiterpenoids Enabled by Combining Metabolic Engineering and Visible‐Light Photocatalysis
Bio-Chem collaboration: Escherichia coli fermentation and visible-light photocatalysis were combined in a complementary way for the first time, to realize the scaffold assembly in a “one-pot” fashion and the subsequent diversifications in a “one-step” manner, respectively, enabling the production of six sesquiterpenoids.
Abstract
The diversification of natural products to expand biologically relevant chemical space for drug discovery can be achieved by combining complementary bioprocessing and chemical transformations. Herein, genetically engineered Escherichia coli fermentation to produce amorphadiene and valencene was combined with metal-free photocatalysis transformations to further access nootkatone, cis-nootkatol and two hydration derivatives. In fermentation, using a closed, anaerobic condition avoided the use of organic overlay, increased the productivity, and simplified the work-up process. Metal-free photocatalysis hydration and allylic C−H oxidation were designed and implemented to make the whole process greener. It was shown that the anti-Markovnikov selectivity of photocatalyzed alkene hydration could be reversed by stereo-electronic and steric effects existing in complex natural products. The combination of bioprocessing and photocatalysis may provide an efficient and greener way to expand the chemical space for pharmaceutical, flavor and fragrance industry.
Directed evolution of a cyclodipeptide synthase with new activities via label-free mass spectrometric screening
DOI: 10.1039/D2SC01637K, Edge Article
A robotic workflow for directed evolution of new enzymatic activities via high-throughput library creation and label-free MS screening.
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Enzymatic Enantioselective anti‐Markovnikov Hydration of Aryl Alkenes
Styrene monooxygenase catalyzed enantioselective anti-Markovnikov hydration of aryl alkenes, rather than epoxidation, was made possible by simply removing oxygen from the reaction mixture. An acid–base mechanism with a carbanion-like intermediate enabled the reaction with excellent anti-Markovnikov regioselectivity.
Abstract
The addition of water to alkenes is an important method for the synthesis of alcohols, but the regioselectivity of acid-catalyzed hydration of terminal alkenes yields secondary alcohols according to Markovnikov's rule, making it difficult to obtain primary alcohols. Here we report a styrene monooxygenase that catalyzes the anti-Markovnikov hydration of the terminal aryl alkenes under anaerobic conditions. This hydration provides primary alcohols in good yields (up to 100 %), excellent anti-Markovnikov regioselectivity (>99 : 1), and good enantiomeric purity (60–83 % ee). Residues Asn46, Asp100, and Asn309 are essential for catalysis suggesting an acid–base mechanism with a carbanion-like intermediate that could account for the anti-Markovnikov regioselectivity. Our work reveals a new enzymatic tool with unusual regioselectivity based on the promiscuous catalytic activity of a monooxygenase.
[ASAP] Remote-Controlled Exchange Rates by Photoswitchable Internal Catalysis of Dynamic Covalent Bonds
Tailoring the squalene-hopene cyclase for stereoconvergent and efficient cationic cyclization cascades
[ASAP] Use of Noncanonical Tyrosine Analogues to Probe Control of Radical Intermediates during Endoperoxide Installation by Verruculogen Synthase (FtmOx1)
Merging the Non‐Natural Catalytic Activity of Lipase and Electrosynthesis: Asymmetric Oxidative Cross‐Coupling of Secondary Amines with Ketones
An asymmetric oxidative cross-coupling strategy that combines the non-natural catalytic activity of lipase with organic electrosynthesis was developed. This unprecedented protocol demonstrates that hydrolase catalysis is compatible with electrosynthesis.
Abstract
We describe the enantioselective oxidative cross-coupling of secondary amines with ketones by combining the non-natural catalytic activity of lipase with electrosynthesis. Various 2,2-disubstituted 3-carbonyl indoles with a stereogenic quaternary carbon center were synthesized from 2-substituted indoles in yields up to 78 % with good enantio- and diastereoselectivities (up to 96 : 4 e.r. and >20 : 1 d.r.). This unprecedented protocol demonstrated that hydrolase catalysis is compatible with electrosynthesis, and the reaction can be carried out in organic solvents with a broad substrate scope and good stereoselectivity. This work provides insights into enzymatic electrosynthesis.
Allosteric rescue of catalytically impaired ATP phosphoribosyltransferase variants links protein dynamics to active-site electrostatic preorganisation
Micelle-guided Morita–Baylis–Hillman reaction of ketones in water
DOI: 10.1039/D2OB00638C, Communication
The Morita–Baylis–Hillman reaction of ketones with electron-deficient alkenes is reported in a micellar medium through co-operative catalysis.
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[ASAP] Tuning Enzyme Thermostability via Computationally Guided Covalent Stapling and Structural Basis of Enhanced Stabilization
Carboxylate Catalyzed Isomerization of β,γ‐Unsaturated N‐Acetylcysteamine Thioesters
The power of carboxylates: Simple carboxylate salts can rival strong amidine bases, such as DBU, in their catalytic power to isomerize β,γ-unsaturated thioesters to corresponding conjugated α,β-unsaturated thioesters. The mechanism involves a rate-determining protonation step!
Abstract
We demonstrate herein the capacity of simple carboxylate salts – tetrametylammonium and tetramethylguanidinium pivalate – to act as catalysts in the isomerization of β,γ-unsaturated thioesters to α,β-unsaturated thioesters. The carboxylate catalysts gave reaction rates comparable to those obtained with DBU, but with fewer side reactions. The reaction exhibits a normal secondary kinetic isotope effect (k 1H/k 1D=1.065±0.026) with a β,γ-deuterated substrate. Computational analysis of the mechanism provides a similar value (k 1H/k 1D=1.05) with a mechanism where γ-reprotonation of the enolate intermediate is rate determining.
Redox- and metal-directed structural diversification in designed metalloprotein assemblies
DOI: 10.1039/D2CC02440C, Communication
Herein we describe a designed protein building block whose self-assembly behaviour is dually gated by the redox state of disulphide bonds and the identity of exogenous metal ions.
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Front Cover: Tetraphenylporphyrin Enters the Ring: First Example of a Complex between Highly Bulky Porphyrins and a Protein (ChemBioChem 14/2022)
Metallotetraphenylporphyrins (metalloTPPs) are exceedingly hydrophobic, rendering them insoluble in aqueous solvents. Upon introduction of the bacterial haem-acquisition protein, HasA, which can incorporate bulky metalloTPPs in its haem-binding pocket, a stable, water-soluble HasA–TPP complex can be formed. This permits the utilisation of metalloTPPs, in the form of a HasA–TPP complex, with biological systems, as antimicrobial agents targeting the critical pathogen Pseudomonas aeruginosa or as biocatalysts. The picture was created by Yuma Shisaka and Mami Yoshimura. More information can be found in the Research Article by O. Shoji et al.
One‐Step Biocatalytic Synthesis of Sustainable Surfactants by Selective Amide Bond Formation
An enzymatic route to commercially important surfactants is presented. A truncated construct of carboxylic acid reductase (CARmm-A) catalyzes amide bond formation between fatty acids and amino alcohols with no esterification observed. The wide substrate scope of the enzyme, co-factor recycling, reaction engineering and up-scaling show the feasibility of this method for synthesis.
Abstract
N-alkanoyl-N-methylglucamides (MEGAs) are non-toxic surfactants widely used as commercial ingredients, but more sustainable syntheses towards these compounds are highly desirable. Here, we present a biocatalytic route towards MEGAs and analogues using a truncated carboxylic acid reductase construct tailored for amide bond formation (CARmm-A). CARmm-A is capable of selective amide bond formation without the competing esterification reaction observed in lipase catalysed reactions. A kinase was implemented to regenerate ATP from polyphosphate and by thorough reaction optimisation using design of experiments, the amine concentration needed for amidation was significantly reduced. The wide substrate scope of CARmm-A was exemplified by the synthesis of 24 commercially relevant amides, including selected examples on a preparative scale. This work establishes acyl-phosphate mediated chemistry as a highly selective strategy for biocatalytic amide bond formation in the presence of multiple competing alcohol functionalities.
Non-native Anionic Ligand Binding and Reactivity in Engineered Variants of the Fe(II)- and α-Ketoglutarate-Dependent Oxygenase, SadA
Retraction Note: The Arabidopsis NOT4A E3 ligase promotes PGR3 expression and regulates chloroplast translation
R.B. Leveson-Goweroooph that would suck so much
Nature Communications, Published online: 20 May 2022; doi:10.1038/s41467-022-30354-z
Retraction Note: The Arabidopsis NOT4A E3 ligase promotes PGR3 expression and regulates chloroplast translation[ASAP] A Nonconventional Archaeal Fluorinase Identified by In Silico Mining for Enhanced Fluorine Biocatalysis
[ASAP] C–C Bond Forming Reactions Enabled by Vitamin B12Opportunities and Challenges
R.B. Leveson-GowerCobalamin enzymes seem tricky but think we will see some cool non-native reactions in the future.
[ASAP] Biocatalytic Carbene Transfer Using Diazirines
A Designed Photoenzyme Promotes Enantioselective [2+2]-Cycloadditions via Triplet Energy Transfer
Cascade Catalysis Through Bifunctional Lipase Metal Biohybrids for the Synthesis of Enantioenriched O‐Heterocycles from Allenes
Hybrid catalysts: Metal nanoparticles are deposited in a polymer-modified protein framework to obtain bifunctional hybrid catalysts that combine the transition metal activities with the host biocatalysts′ activation mode. A tailor-made lipase-silver nanobiohybrid is successfully exploited in a cascade design where a racemic allenic acetate is transformed to an enantioenriched dihydrofuran via a sequential hydrolytic kinetic resolution and a cycloisomerization.
Abstract
Lipase/metal nanobiohybrids, generated by growth of silver or gold nanoparticles on protein matrixes are used as highly effective dual-activity heterogeneous catalysts for the production of enantiomerically enriched 2,5-dihydrofurans from allenic acetates in a one-pot cascade process combining a lipase-mediated hydrolytic kinetic resolution with a metal-catalyzed allene cycloisomerization. Incorporating a novel strategy based on enzyme-polymer bioconjugates in the nanobiohybrid preparation enables excellent conversions in the process. Candida antarctica lipase B (CALB) in combination with a dextran-based polymer modifier (DexAsp) proved to be most efficient when merged with silver nanoparticles. A range of hybrid materials were produced, combining Ag or Au metals with Thermomyces lanuginosus lipase (TLL) or CALB and its DexAsp or polyethyleneimine polymer bioconjugates. The wider applicability of the biohybrids is demonstrated by their use in allenic alcohol cyclizations, where a variety of dihydrofurans are obtained using a CALB/gold nanomaterial. These results underline the potential of the nanobiohybrid catalysis as promising approach to intricate one-pot synthetic strategies.
Biocatalytic Enantioselective β‐Hydroxylation of Unactivated C−H Bonds in Aliphatic Carboxylic Acids
Directed evolution rendered P450BSβ capable of β-hydroxylating unactivated C−H bonds in aliphatic carboxylic acids with broad substrate scope and excellent chemo-, regio-, and enantioselectivity. The crystal structure of the evolved variant rationalizes the improved reactivity and selectivity. This study demonstrates the potential of exploring biocatalysts to fulfill reactions that are otherwise elusive with chemical strategies.
Abstract
Catalytic selective hydroxylation of unactivated aliphatic (sp3) C−H bonds without a directing group represents a formidable task for synthetic chemists. Through directed evolution of P450BSβ hydroxylase, we realize oxyfunctionalization of unactivated C−H bonds in a broad spectrum of aliphatic carboxylic acids with varied chain lengths, functional groups and (hetero-)aromatic moieties in a highly chemo-, regio- and enantioselective fashion (>30 examples, Cβ/Cα>20 : 1, >99 % ee). The X-ray structure of the evolved variant, P450BSβ-L78I/Q85H/G290I, in complex with palmitic acid well rationalizes the experimentally observed regio- and enantioselectivity, and also reveals a reduced catalytic pocket volume that accounts for the increased reactivity with smaller substrates. This work showcases the potential of employing a biocatalyst to enable a chemical transformation that is particularly challenging by chemical methods.
[ASAP] Kinetic, Inhibition, and Structural Characterization of a Malonate Semialdehyde Decarboxylase-like Protein from Calothrix sp. PCC 6303: A Gateway to the non-Pro1 Tautomerase Superfamily Members
[ASAP] Stereodivergent Synthesis of Epoxides and Oxazolidinones via the Halohydrin Dehalogenase-Catalyzed Desymmetrization Strategy
Unlocking New Reactivities in Enzymes by Iminium Catalysis
This Minireview summarizes the different strategies used in the design and engineering of novel enzymes that accommodate iminium catalysis. The advantages and challenges of developing enzymes for this catalysis mode are discussed. Recent developments in iminium biocatalysis showcase the tremendous power of combining chemomimetic biocatalyst design and directed evolution to create useful biocatalysts for new-to-nature transformations.
Abstract
The application of biocatalysis in conquering challenging synthesis requires the constant input of new enzymes. Developing novel biocatalysts by absorbing catalysis modes from synthetic chemistry has yielded fruitful new-to-nature enzymes. Organocatalysis was originally bio-inspired and has become the third pillar of asymmetric catalysis. Transferring organocatalytic reactions back to enzyme platforms is a promising approach for biocatalyst creation. Herein, we summarize recent developments in the design of novel biocatalysts that adopt iminium catalysis, a fundamental branch in organocatalysis. By repurposing existing enzymes or constructing artificial enzymes, various biocatalysts for iminium catalysis have been created and optimized via protein engineering to promote valuable abiological transformations. Recent advances in iminium biocatalysis illustrate the power of combining chemomimetic biocatalyst design and directed evolution to generate useful new-to-nature enzymes.
[ASAP] “Multiagent” Screening Improves Directed Enzyme Evolution by Identifying Epistatic Mutations
Organocatalytic stereoselective cyanosilylation of small ketones
Nature, Published online: 04 May 2022; doi:10.1038/s41586-022-04531-5
The development of confined organocatalysts for the enantioselective cyanosilylation of small, unbiased substrates, including 2-butanone, is shown to lead to catalysts that are as selective as enzymes, with excellent levels of control.