R.B. Leveson-Gower

ATP phosphoribosyltransferase catalyses the first step of histidine biosynthesis and is controlled via a complex allosteric mechanism where the regulatory protein HisZ enhances catalysis by the catalytic protein HisGS while mediating allosteric inhibition by histidine. Activation by HisZ was proposed to position HisGS Arg56 to stabilise departure of the pyrophosphate leaving group. Here we report active-site mutants of HisGS with impaired reaction chemistry which can be allosterically restored by HisZ despite the HisZ:HisGS interface lying ~20-Å away from the active site. MD simulations indicate HisZ binding constrains the dynamics of HisGS to favour a preorganised active site where both Arg56 and Arg32 are poised to stabilise leaving-group departure in WT-HisGS. In the Arg56Ala-HisGS mutant, HisZ modulates Arg32 dynamics so that it can partially compensate for the absence of Arg56. These results illustrate how remote protein:protein interactions translate into catalytic resilience by restoring damaged electrostatic preorganisation at the active site.

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.

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.

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.

Mononuclear non-heme Fe(II)- and -ketoglutarate dependent oxygenases (FeDOs) catalyze site-selective C-H hydroxylation. Variants of these enzymes in which a conserved Asp/Glu residue in the Fe(II)-binding facial triad is replaced by Ala/Gly can, in some cases, bind various anionic ligands and catalyze non-native chlorination and bromination reactions. In this study, we explore the binding of different anions to a FeDO facial triad variant, SadX, and the effects of that binding on HO• vs. X• rebound. We establish that chloride and bromide not only enable non-native halogenation reactions but that all anions investigated, including azide, cyanate, formate, and fluoride, significantly accelerate and influence the site selectivity of SadX hydroxylation catalysis. Azide and cyanate also lead to the formation of products resulting from N3•, NCO•, and OCN• rebound. While fluoride rebound is not observed, the rate acceleration provided by this ligand led us to calculate barriers for HO• and F• rebound from a putative Fe(III)(OH)(F) intermediate. These calculations suggest that the lack of fluorination is due to the relative barriers of the HO• and F• rebound transition states rather than an inaccessible barrier for F• rebound. Together, these results improve our understanding of FeDO facial triad variant tolerance of different anionic ligands, their ability to promote rebound involving those ligands, and inherent rebound preferences relative to HO• that will aid efforts to develop non-native catalysis using these enzymes.

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

The ability to programme new modes of catalysis into proteins would allow the development of enzyme families with functions beyond those found in nature. To this end, genetic code expansion methodology holds particular promise, as it allows the site-selective introduction of new functional elements into proteins as non-canonical amino acid side chains. Here, we exploit an expanded genetic code to develop a photoenzyme that operates via triplet energy transfer catalysis, a versatile mode of reactivity in organic synthesis that is currently not accessible to biocatalysis. Installation of a genetically encoded photosensitiser into the beta-propeller scaffold of DA_20_00 converts a de novo Diels-Alderase into a photoenzyme for [2+2]-cycloadditions (EnT1.0). Subsequent development and implementation of a platform for photoenzyme evolution afforded an efficient and enantioselective enzyme (EnT1.3, up to 99% e.e.) that can promote selective cycloadditions that have proven challenging to achieve with small molecule catalysts. EnT1.3 performs >300 turnovers and, in contrast to small molecule photocatalysts, can operate effectively under aerobic conditions. A 1.7 Å resolution X-ray crystal structure of an EnT1.3-product complex shows how multiple functional components work in synergy to promote efficient and selective photocatalysis. This study opens the door to a wealth of new excited-state chemistry in protein active sites and establishes the framework for developing a new generation of evolvable photocatalysts with efficiencies and specificities akin to natural enzymes.

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.

Directed evolution rendered P450_BSβ 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 P450_BSβ 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, P450_BSβ-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.

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.

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.

A noncovalent photoswitch for regulating enzyme activity was synthesized based on a polymeric inhibitor-encapsulation method. This method noncovalently anchors an azobenzene-modified inhibitor to the active site of the encapsulated enzyme, allowing reversible control of the enzymatic activity using light. This approach provides a promising strategy to regulate the activity of the enzymes without genetic mutation nor chemical modification of enzyme.

Abstract

Photochemical regulation provides a promising approach for controlling enzyme activity on demand owing to its high spatiotemporal resolution. However, reversible regulation of the enzyme activity by light usually requires genetic mutations and covalent modifications of the target enzymes, which may lead to irreversible changes in the enzyme structure and subsequent loss of the enzymatic activity. Herein, we have developed a novel strategy based on a polymeric inhibitor-encapsulated enzyme, which noncovalently anchors the azobenzene-modified inhibitors to the enzyme active site, thereby achieving reversible control of the activity of native enzymes using light. As neither genetic mutation nor chemical modification of enzymes is required for this method, negligible loss of the enzymatic activity was observed for the encapsulated enzymes compared to their native counterparts. Thus, this approach has demonstrated a promising strategy for achieving reversible regulation of the activity of native enzymes.

Biocatalytic carbene transfer from diazo compounds is a versatile strategy in asymmetric synthesis. However, the limited pool of stable diazo compounds constrains the variety of accessible products. To overcome this restriction, we have engineered variants of Aeropyrum pernix protoglobin (ApePgb) that use diazirines as carbene precursors. While the enhanced stability of diazir- ines relative to their diazo isomers enables access to a diverse array of carbenes, they have previously resisted catalytic activation. Our engineered ApePgb variants represent the first example of catalysts for selective carbene transfer from these species at room temperature. The structure of an ApePgb variant, determined by microcrystal electron diffraction (MicroED), reveals that evolution has enhanced access to the heme active site to facilitate this new-to-nature catalysis. Using readily prepared aryl diazirines as model substrates, we demonstrate the application of these highly-stable carbene precursors in biocatalytic cyclopropanation, N–H insertion, and Si–H insertion reactions.

The conversion of carboxylic acids to thioesters is a key step in the biosynthesis of natural products, resulting in activation of the acyl groups for subsequent reactions, e.g. acylation of nucleophiles including carbon-carbon bond formation. For example, thioesters of Coenzyme A (CoA-SH; e.g. acetyl-S-CoA) are intermediates in many metabolic pathways, and are increasingly recognised as important cofactors for epigenetic post-translational modifications, such as N-, O- and S-acylations of proteins. However, the limited availability of a broad range of structurally diverse thioesters has limited their wider exploitation in biochemistry, cell biology and biotechnology. Furthermore, the high cost of CoA-SH impairs its use in stoichiometric quantities. To address these challenges we show that the adenylation (A-) domain of the carboxylic acid reductase (CAR) from Segniliparus rugosus (CARsr-A) can function as a broad spectrum acyl-S-CoA synthetase, to generate acyl-S-CoA intermediates from a wide range of carboxylic acids. In addition, CARsr-A was able to generate thioesters from structurally simpler thiols such as pantetheine. The resulting thioesters were then used as substrates for acyltransferases to synthesise a wide range of amides, including the more difficult to prepare, but pharmaceutically relevant aryl amides. Importantly, CoA-SH is recycled during the reaction and can be used in sub-stoichiometric quantities. This approach has also been applied to acylate a histone peptide H4-20 with a range of carboxylic acids, including non-natural chemical labels, by employing a lysine acetyltransferase (HATp300). Overall, this combination of a broad spectrum biocatalyst for thioester synthesis, together with in-situ CoA-SH recycling, provides a generic platform for thioester-dependent cell-free synthesis, with potential applications beyond amide bond formation.

Nature Catalysis, Published online: 02 May 2022; doi:10.1038/s41929-022-00777-4

Engineering enzymes to perform new-to-nature reactions can address long-standing challenges in synthetic chemistry. Now a ketoreductase has been evolved to undergo a photoinduced single-electron-transfer pathway, thereby achieving an enantioselective Giese-type radical conjugate addition that yields α-chiral esters.

Enzymes continue to gain recognition as valuable tools in synthetic chemistry as they enable transformations, which elude conventional organochemical approaches. As such, the progressing expansion of the biocatalytic arsenal has introduced unprecedented opportunities for new synthetic strategies and retrosynthetic disconnections. As a result, enzymes have found a solid foothold in modern natural product synthesis for applications ranging from the generation of early chiral synthons to endgame transformations, convergent synthesis and cascade reactions for the rapid construction of molecular complexity. As a primer to the state-of-the-art concerning strategic uses of enzymes in natural product synthesis and the underlying concepts, this review highlights selected recent literature examples (covering 2020 to April 2022), which make a strong case for the admission of enzymatic methodologies into the standard repertoire for complex small molecule synthesis.

Nature, Published online: 27 April 2022; doi:10.1038/s41586-022-04599-z

Untreated, postconsumer-PET from 51 different thermoformed products can all be almost completely degraded by FAST-PETase in 1 week and PET can be resynthesized from the recovered monomers, demonstrating recycling at the industrial scale.

In this study, we engineer a variant of the flavin-dependent halogenase RebH that catalyzes site- and atroposelective halogenation of 3-aryl-4(3H)-quinazolinones via kinetic or dynamic kinetic resolution. The required directed evolution uses a combination of random and site-saturation mutagenesis, substrate walking using two probe substrates, and a two-tiered screening approach involving analysis of variant conversion and then enantioselectivity of improved variants. The resulting variant, 3-T, provides >99:1 e.r. for the (M)-atropisomer of the major brominated product, 25-fold improved conversion, and 91-fold improved site-selectivity relative to the parent enzyme on the probe substrate used in the final rounds of evolution. This high activity and selectivity translates well to several additional substrates with varied steric and electronic properties. Computational modeling and docking simulations are used to rationalize the effects of key mutations on substrate scope and site- and atroposelectivity. Given the range of substrates that have been used for atroposelective synthesis via electrophilic halogenation, these results suggest that FDHs could find many additional applications for atroposelective catalysis. More broadly, this study highlights how RebH can be engineered to accept structurally diverse substrates that enable its use for enantioselective catalysis.