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Unspecific Peroxygenases (UPOs) have emerged as robust biocatalysts for selective oxygenation reactions, as they are easily produced at scale and require only hydrogen peroxide as the external oxidant. UPOs can catalyze the oxygenation of the primary benzylic carbons of toluenes to give alcohol, aldehyde and carboxylic acid products. They can also catalyze hydroxylation at the benzylic position of ethylbenzenes, and the subsequent oxidation of the secondary alcohols to ketones. In this study, we have investigated factors that affect the balance of products in UPO‐catalyzed benzylic oxygenations using a range of functionalised toluenes and ethyl benzenes, and a UPO from Agrocybe aegerita (rAaeUPO‐PaDa‐I‐H variant). The product distribution is dependent upon a mixture of steric and electronic effects and, in selected cases, controlling the reaction conditions permits products from each product series to be generated chemoselectively. In this way, electron poor toluenes were converted directly into carboxylic acids in isolated yields of 36–99 % on preparative scale. [ABSTRACT FROM AUTHOR]

Unspecific Peroxygenases (UPOs) are increasingly significant enzymes for selective oxygenations as they are stable, highly active and catalyze their reactions at the expense of only hydrogen peroxide as the oxidant. Their structural similarity to chloroperoxidase (CPO) means that UPOs can also catalyze halogenation reactions based upon the generation of hypohalous acids from halide and H2O2. Here we show that the halogenation and oxygenation modes of a UPO can be stimulated at different pH values. Using simple aromatic compounds such as thymol, we show that, at a pH of 3.0 and 6.0, either brominated or oxygenated products respectively are produced. Preparative 100 mg scale transformations of substrates were performed with 60–72 % isolated yields of brominated products obtained. A one‐pot bromination‐oxygenation cascade reaction on 4‐ethylanisole, in which the pH was adjusted from 3.0 to 6.0 at the halfway stage, yielded sequentially brominated and oxygenated products 1‐(3‐bromo‐4‐methoxyphenyl)ethyl alcohol and 3‐bromo‐4‐methoxy acetophenone with 82 % combined conversion. These results identify UPOs as an unusual example of a biocatalyst that is tunable for entirely different chemical reactions, dependent upon the reaction conditions. [ABSTRACT FROM AUTHOR]

Silyl ethers fulfil a fundamental role in synthetic organic chemistry as protecting groups and their selective cleavage is an important factor in their application. We present here for the first time two enzymes, SilE−R and SilE−S, which are able to hydrolyse silyl ethers. They belong to the stress‐response dimeric A/B barrel domain (DABB) family and are able to cleave the Si−O bond with opposite enantiopreference. Silyl ethers containing aromatic, cyclic or aliphatic alcohols and, depending on the alcohol moiety, silyl functions as large as TBDMS are accepted. The X‐ray crystal structure of SilE−R, determined to a resolution of 1.98 Å, in combination with mutational studies, revealed an active site featuring two histidine residues, H8 and H79, which likely act synergistically as nucleophile and Brønsted base in the hydrolytic mechanism, which has not previously been described for enzymes. Although the natural function of SilE−R and SilE−S is unknown, we propose that these 'silyl etherases' may have significant potential for synthetic applications. [ABSTRACT FROM AUTHOR]

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Native amine dehydrogenases offer sustainable access to chiral amines, so the search for scaffolds capable of converting more diverse carbonyl compounds is required to reach the full potential of this alternative to conventional synthetic reductive aminations. Here we report a multidisciplinary strategy combining bioinformatics, chemoinformatics and biocatalysis to extensively screen billions of sequences in silico and to efficiently find native amine dehydrogenases features using computational approaches. In this way, we achieve a comprehensive overview of the initial native amine dehydrogenase family, extending it from 2,011 to 17,959 sequences, and identify native amine dehydrogenases with non-reported substrate spectra, including hindered carbonyls and ethyl ketones, and accepting methylamine and cyclopropylamine as amine donor. We also present preliminary model-based structural information to inform the design of potential (R)-selective amine dehydrogenases, as native amine dehydrogenases are mostly (S)-selective. This integrated strategy paves the way for expanding the resource of other enzyme families and in highlighting enzymes with original features. Sustainable chemistry can benefit from biocatalysis, but a high diversity of enzymes is needed. Here, the authors screen billions of protein sequences to provide an overview of the native amine dehydrogenase family for amine synthesis. [ABSTRACT FROM AUTHOR]

The immobilisation of artificial metalloenzymes (ArMs) holds promise for the implementation of new biocatalytic reactions. We present the synthesis of cross-linked artificial metalloenzyme aggregates (CLArMAs) with excellent recyclability, as an alternative to carrier-based immobilisation strategies. Furthermore, iron-siderophore supramolecular anchoring facilitates redox-triggered cofactor release, enabling CLArMAs to be recharged with alternative cofactors for diverse selectivity. [ABSTRACT FROM AUTHOR]

Macrocyclic and medium‐sized ring ketones, lactones and lactams can all be made from common acryloyl imide starting materials through divergent, one‐pot cascade ring‐expansion reactions. Following either conjugate addition with an amine or nitromethane, or osmium(VIII)‐catalysed dihydoxylation, rearrangement through a four‐atom ring expansion takes place spontaneously to form the ring expanded products. A second ring expansion can also be performed following a second iteration of imide formation and alkene functionalisation/ring expansion. In the dihydroxylation series, three‐ or four‐atom ring expansion can be performed selectively, depending on whether the reaction is under kinetic or thermodynamic control. [ABSTRACT FROM AUTHOR]

The NADPH‐dependent imine reductase from Ajellomyces dermatitidis (AdRedAm) catalyzes the reductive amination of certain ketones with amine donors supplied in an equimolar ratio. The structure of AdRedAm has been determined in three forms. The first form, which belongs to space group P3121 and was refined to 2.01 Å resolution, features two molecules (one dimer) in the asymmetric unit in complex with the redox‐inactive cofactor NADPH4. The second form, which belongs to space group C21 and was refined to 1.73 Å resolution, has nine molecules (four and a half dimers) in the asymmetric unit, each complexed with NADP+. The third form, which belongs to space group P3121 and was refined to 1.52 Å resolution, has one molecule (one half‐dimer) in the asymmetric unit. This structure was again complexed with NADP+ and also with the substrate 2,2‐difluoroacetophenone. The different data sets permit the analysis of AdRedAm in different conformational states and also reveal the molecular basis of stereoselectivity in the transformation of fluorinated acetophenone substrates by the enzyme. [ABSTRACT FROM AUTHOR]

Deoxypodophyllotoxin synthase (DPS) is a 2-oxoglutarate (2-OG) dependent non-heme iron (II) dioxygenase that catalyzes the stereoselective ring-closing carbon-carbon bond formation of deoxypodophyllotoxin from the aryllignan (-)-yatein. Deoxypodophyllotoxin is a precursor of topoisomerase II inhibitors, which are on the World Health Organization's list of essential medicines. Previous work has shown that DPS can accept a range of substrates, indicating it has potential in biocatalytic processes for the formation of diverse polycyclic aryllignans. Recent X-ray structures of the enzyme reveal possible roles for amino acid side chains in substrate recognition and mechanism, although a mutational analysis of DPS was not performed. Here, we present a structure of DPS at an improved resolution of 1.41 Å, in complex with the buffer molecule, Tris, coordinated to the active site iron atom. The structure has informed a mutational analysis of DPS, which suggests a role for a D224-K187 salt bridge in maintaining substrate interactions and a catalytic role for H165, perhaps as the base for the proton abstraction at the final rearomatization step. This work improves our understanding of specific residues' contributions to the DPS mechanism and can inform future engineering of the enzyme mechanism and substrate scope for the development of a versatile biocatalyst. [ABSTRACT FROM AUTHOR]

A lyophilized preparation of an unspecific peroxygenase variant from Agrocybe aegerita (rAaeUPO‐PaDa‐I‐H) is a highly effective catalyst for the oxygenation of a diverse range of N‐heterocyclic compounds. Scalable biocatalytic oxygenations (27 preparative examples, ca. 100 mg scale) have been developed across a wide range of substrates, including alkyl pyridines, bicyclic N‐heterocycles and indoles. H2O2 is the only stoichiometric oxidant needed, without auxiliary electron transport proteins, which is key to the practicality of the method. Reaction outcomes can be altered depending on whether hydrogen peroxide was delivered by syringe pump or through in situ generation using an alcohol oxidase from Pichia pastoris (PpAOX) and methanol as a co‐substrate. Good synthetic yields (up to 84 %), regioselectivity and enantioselectivity (up to 99 % ee) were observed in some cases, highlighting the promise of UPOs as practical, versatile and scalable oxygenation biocatalysts. [ABSTRACT FROM AUTHOR]

A lyophilized preparation of an unspecific peroxygenase variant from Agrocybe aegerita (rAaeUPO‐PaDa‐I‐H) is a highly effective catalyst for the oxygenation of a diverse range of N‐heterocyclic compounds. Scalable biocatalytic oxygenations (27 preparative examples, ca. 100 mg scale) have been developed across a wide range of substrates, including alkyl pyridines, bicyclic N‐heterocycles and indoles. H2O2 is the only stoichiometric oxidant needed, without auxiliary electron transport proteins, which is key to the practicality of the method. Reaction outcomes can be altered depending on whether hydrogen peroxide was delivered by syringe pump or through in situ generation using an alcohol oxidase from Pichia pastoris (PpAOX) and methanol as a co‐substrate. Good synthetic yields (up to 84 %), regioselectivity and enantioselectivity (up to 99 % ee) were observed in some cases, highlighting the promise of UPOs as practical, versatile and scalable oxygenation biocatalysts. [ABSTRACT FROM AUTHOR]

Native amine dehydrogenases (nat-AmDHs) catalyze the (S)-stereoselective reductive amination of various ketones and aldehydes in the presence of high concentrations of ammonia. Based on the structure of CfusAmDH from Cystobacter fuscus complexed with Nicotinamide adenine dinucleotide phosphate (NADP+) and cyclohexylamine, we previously hypothesized a mechanism involving the attack at the electrophilic carbon of the carbonyl by ammonia followed by delivery of the hydride from the reduced nicotinamide cofactor on the re-face of the prochiral ketone. The direct reduction of carbonyl substrates into the corresponding alcohols requires a similar active site architecture and was previously reported as a minor side reaction of some native amine dehydrogenases and variants. Here we describe the ketoreductase (KRED) activity of a set of native amine dehydrogenases and variants, which proved to be significant in the absence of ammonia in the reaction medium but negligible in its presence. Conducting this study on a large set of substrates revealed the heterogeneity of this secondary ketoreductase activity, which was dependent upon the enzyme/substrate pairs considered. In silico docking experiments permitted the identification of some relationships between ketoreductase activity and the structural features of the enzymes. Kinetic studies of MsmeAmDH highlighted the superior performance of this native amine dehydrogenases as a ketoreductase but also its very low activity towards the reverse reaction of alcohol oxidation. [ABSTRACT FROM AUTHOR]

Native Amine Dehydrogenases (nat‐AmDHs) are NAD(P)H‐enzymes performing reductive amination, mainly active towards small aliphatic aldehydes and cyclic ketones, due to active site volumes limited by the presence of several bulky amino acids. Herein, inspired by the diversity of residues at these positions among the family, we report the implementation of mutations F140A and W145A in CfusAmDH and their transposition in nine other members. Moderate to high conversions were obtained with substrates not accepted by the native enzymes, notably n‐alkylaldehydes (44.6 %–99.5 % for hexanal to nonanal) and n‐alkylketones (16.0 %–53.7 % for hexan‐2‐one to nonan‐2‐one) with retention of excellent (S)‐enantioselectivity (>99 % ee). Complementary to the reported (R)‐selective AmDHs, the promising mutant CfusAmDH−W145A was further characterized for its synthetic potential. Crystal structure resolution and molecular dynamics gave insights into the cofactor and substrate specificity and the whole structural dynamics, thus providing keys for mutagenesis work on this enzyme family. [ABSTRACT FROM AUTHOR]

H2O2‐driven enzymes are of great interest for industrial biotransformations. Herein, we show for the first time that oxalate oxidase (OXO) is an efficient in situ source of H2O2 for one of these biocatalysts, which is known as unspecific peroxygenase (UPO). OXO is reasonably robust, produces only CO2 as a by‐product and uses oxalate as a cheap sacrificial electron donor. UPO has significant potential as an industrial catalyst for selective C−H oxyfunctionalisations, as we confirm herein by testing a diverse drug panel using miniaturised high‐throughput assays and mass spectrometry. 33 out of 64 drugs were converted in 5 μL‐scale reactions by the UPO with OXO (conversion >70 % for 11 drugs). Furthermore, oxidation of the drug tolmetin was achieved on a 50 mg scale (TONUPO 25 664) with 84 % yield, which was further improved via enzyme immobilization. This one‐pot approach ensures adequate H2O2 levels, enabling rapid access to industrially relevant molecules that are difficult to obtain by other routes. [ABSTRACT FROM AUTHOR]

H2O2‐driven enzymes are of great interest for industrial biotransformations. Herein, we show for the first time that oxalate oxidase (OXO) is an efficient in situ source of H2O2 for one of these biocatalysts, which is known as unspecific peroxygenase (UPO). OXO is reasonably robust, produces only CO2 as a by‐product and uses oxalate as a cheap sacrificial electron donor. UPO has significant potential as an industrial catalyst for selective C−H oxyfunctionalisations, as we confirm herein by testing a diverse drug panel using miniaturised high‐throughput assays and mass spectrometry. 33 out of 64 drugs were converted in 5 μL‐scale reactions by the UPO with OXO (conversion >70 % for 11 drugs). Furthermore, oxidation of the drug tolmetin was achieved on a 50 mg scale (TONUPO 25 664) with 84 % yield, which was further improved via enzyme immobilization. This one‐pot approach ensures adequate H2O2 levels, enabling rapid access to industrially relevant molecules that are difficult to obtain by other routes. [ABSTRACT FROM AUTHOR]

Chiral amine diastereomers are ubiquitous in pharmaceuticals and agrochemicals1, yet their preparation often relies on low-efficiency multi-step synthesis2. These valuable compounds must be manufactured asymmetrically, as their biochemical properties can differ based on the chirality of the molecule. Herein we characterize a multifunctional biocatalyst for amine synthesis, which operates using a mechanism that is, to our knowledge, previously unreported. This enzyme (EneIRED), identified within a metagenomic imine reductase (IRED) collection3 and originating from an unclassified Pseudomonas species, possesses an unusual active site architecture that facilitates amine-activated conjugate alkene reduction followed by reductive amination. This enzyme can couple a broad selection of α,β-unsaturated carbonyls with amines for the efficient preparation of chiral amine diastereomers bearing up to three stereocentres. Mechanistic and structural studies have been carried out to delineate the order of individual steps catalysed by EneIRED, which have led to a proposal for the overall catalytic cycle. This work shows that the IRED family can serve as a platform for facilitating the discovery of further enzymatic activities for application in synthetic biology and organic synthesis.A biocatalytic enzyme originating from bacteria, EneIRED, facilitates amine-activated conjugate alkene reduction followed by reductive amination, efficiently preparing chiral amine diastereomers, which are commonly used in pharmaceuticals and agrochemicals. [ABSTRACT FROM AUTHOR]

Imine reductases (IREDs) offer biocatalytic routes to chiral amines and have a natural preference for the NADPH cofactor. In previous work, we reported enzyme engineering of the (R)‐selective IRED from Myxococcus stipitatus (NADH‐IRED‐Ms) yielding a NADH‐dependent variant with high catalytic efficiency. However, no IRED with NADH specificity and (S)‐selectivity in asymmetric reductions has yet been reported. Herein, we applied semi‐rational enzyme engineering to switch the selectivity of NADH‐IRED‐Ms. The quintuple variant A241V/H242Y/N243D/V244Y/A245L showed reverse stereopreference in the reduction of the cyclic imine 2‐methylpyrroline compared to the wild‐type and afforded the (S)‐amine product with >99 % conversion and 91 % enantiomeric excess. We also report the crystal‐structures of the NADPH‐dependent (R)‐IRED‐Ms wild‐type enzyme and the NADH‐dependent NADH‐IRED‐Ms variant and molecular dynamics (MD) simulations to rationalize the inverted stereoselectivity of the quintuple variant. [ABSTRACT FROM AUTHOR]

A chemical method for the treatment of harmful halogenated compounds that has recently become of interest is the reductive dehalogenation of carbon‐halogen bonds. In the case of a fluorine atom, this process is called hydrodefluorination. While many transition metal‐based approaches now exist to reductively defluorinate aromatic fluoroarenes, the cleavage of C−F bonds in aliphatic compounds is not so well‐developed. Here we propose a biocatalytic approach exploiting a promiscuous activity exhibited by transaminases (TAs). Hence, a series of α‐fluoroketones have been defluorinated with excellent conversions using Chromobacterium violaceum and Arthrobacter sp. TAs under mild conditions and in aqueous medium, using a stoichiometric amount of an amine (e. g. 2‐propylamine) as reagent and formally releasing its oxidized form (e. g. acetone), with ammonia and hydrogen fluoride as by‐products. It is also demonstrated that this process can be performed in a regio‐ or stereoselective fashion. [ABSTRACT FROM AUTHOR]

Controlling the selectivity of a chemical reaction with external stimuli is common in thermal processes, but rare in visible‐light photocatalysis. Here we show that the redox potential of a carbon nitride photocatalyst (CN‐OA‐m) can be tuned by changing the irradiation wavelength to generate electron holes with different oxidation potentials. This tuning was the key to realizing photo‐chemo‐enzymatic cascades that give either the (S)‐ or the (R)‐enantiomer of phenylethanol. In combination with an unspecific peroxygenase from Agrocybe aegerita, green light irradiation of CN‐OA‐m led to the enantioselective hydroxylation of ethylbenzene to (R)‐1‐phenylethanol (99 % ee). In contrast, blue light irradiation triggered the photocatalytic oxidation of ethylbenzene to acetophenone, which in turn was enantioselectively reduced with an alcohol dehydrogenase from Rhodococcus ruber to form (S)‐1‐phenylethanol (93 % ee). [ABSTRACT FROM AUTHOR]

Controlling the selectivity of a chemical reaction with external stimuli is common in thermal processes, but rare in visible‐light photocatalysis. Here we show that the redox potential of a carbon nitride photocatalyst (CN‐OA‐m) can be tuned by changing the irradiation wavelength to generate electron holes with different oxidation potentials. This tuning was the key to realizing photo‐chemo‐enzymatic cascades that give either the (S)‐ or the (R)‐enantiomer of phenylethanol. In combination with an unspecific peroxygenase from Agrocybe aegerita, green light irradiation of CN‐OA‐m led to the enantioselective hydroxylation of ethylbenzene to (R)‐1‐phenylethanol (99 % ee). In contrast, blue light irradiation triggered the photocatalytic oxidation of ethylbenzene to acetophenone, which in turn was enantioselectively reduced with an alcohol dehydrogenase from Rhodococcus ruber to form (S)‐1‐phenylethanol (93 % ee). [ABSTRACT FROM AUTHOR]

The biocatalytic asymmetric synthesis of amines from carbonyl compounds and amine precursors presents an important advance in sustainable synthetic chemistry. Oxidoreductases (ORs) that catalyze the NAD(P)H‐dependent reductive amination of carbonyl compounds directly to amines using amine donors present advantages complementary to those of amine transaminases (ATAs) with respect to selectivity, stability and substrate scope. Indeed some ORs accept alkyl and aryl amines as reaction partners enabling access to chiral secondary amine products that are not directly accessible using ATAs. Moreover, superior atom economy can usually be achieved as no sacrificial amines are required as with ATAs. In recent years a number of ORs that apparently catalyze both imine formation and imine reduction in the reductive amination of carbonyls has been identified using structure informed protein engineering, sequence analysis from natural biodiversity and increasingly a mixture of both. In this review we summarize the development of such enzymes from the engineering of amino acid dehydrogenases (AADHs) and opine dehydrogenases (OpDHs) to become amine dehydrogenases (AmDHs), which are active toward ketones devoid of any requisite carboxylate and/or amine functions, through to the discovery of native AmDHs and reductive aminases (RedAms), and the engineering of all of these scaffolds for improved or altered activity. Structural and mechanistic studies have revealed similarities, but also differences in the determinants of substrate binding and mechanism in the enzymes. The survey reveals that a complementary approach to enzyme discovery that utilizes both natural genetic resources and engineering can be combined to deliver biocatalysts that have significant potential for the industrial synthesis of chiral amines. [ABSTRACT FROM AUTHOR]

The synthesis of chiral amines is of central importance to pharmaceutical chemistry, and the inclusion of fluorine atoms in drug molecules can both increase potency and slow metabolism. Optically enriched β‐fluoroamines can be obtained by the kinetic resolution of racemic amines using amine transaminases (ATAs), but yields are limited to 50 %, and also secondary amines are not accessible. In order to overcome these limitations, we have applied NADPH‐dependent reductive aminase enzymes (RedAms) from fungal species to the reductive amination of α‐fluoroacetophenones with ammonia, methylamine and allylamine as donors, to yield β‐fluoro primary or secondary amines with >90 % conversion and between 85 and 99 % ee. In addition, the effect of the progressive introduction of fluorine atoms to the α‐position of the acetophenone substrate reveals the effect of mono‐, di‐ and tri‐fluorination on the proportion of amine and alcohol in product mixtures, shedding light on the promiscuous ability of imine reductase (IRED)‐type dehydrogenases to reduce fluorinated acetophenones to alcohols. [ABSTRACT FROM AUTHOR]

Having identified inconsistencies when repeating literature examples of photochemical transformations and difficulties recreating experimental setups, we devised several criteria that an ideal lab‐scale reactor should achieve. Herein, we introduce a versatile photoreactor for high‐throughput screening, preparative‐scale batch reactions and continuous processing, all with a single light source. The reactor utilizes interchangeable arrays of pseudo‐monochromatic high‐power LEDs in a range of synthetically useful wavelengths, combined with excellent temperature control. Moreover, light intensity can be modulated in an accurate and straightforward manner. This system has subsequently been tested on a range of literature methodologies. [ABSTRACT FROM AUTHOR]

A tandem enzymatic strategy to enhance the scope of C‐alkylation of small molecules via the in situ formation of S‐adenosyl methionine (SAM) cofactor analogues is described. A solvent‐exposed channel present in the SAM‐forming enzyme SalL tolerates 5′‐chloro‐5′‐deoxyadenosine (ClDA) analogues modified at the 2‐position of the adenine nucleobase. Coupling SalL‐catalyzed cofactor production with C‐(m)ethyl transfer to coumarin substrates catalyzed by the methyltransferase (MTase) NovO forms C‐(m)ethylated coumarins in superior yield and greater substrate scope relative to that obtained using cofactors lacking nucleobase modifications. Establishing the molecular determinants that influence C‐alkylation provides the basis to develop a late‐stage enzymatic platform for the preparation of high value small molecules. [ABSTRACT FROM AUTHOR]

A tandem enzymatic strategy to enhance the scope of C‐alkylation of small molecules via the in situ formation of S‐adenosyl methionine (SAM) cofactor analogues is described. A solvent‐exposed channel present in the SAM‐forming enzyme SalL tolerates 5′‐chloro‐5′‐deoxyadenosine (ClDA) analogues modified at the 2‐position of the adenine nucleobase. Coupling SalL‐catalyzed cofactor production with C‐(m)ethyl transfer to coumarin substrates catalyzed by the methyltransferase (MTase) NovO forms C‐(m)ethylated coumarins in superior yield and greater substrate scope relative to that obtained using cofactors lacking nucleobase modifications. Establishing the molecular determinants that influence C‐alkylation provides the basis to develop a late‐stage enzymatic platform for the preparation of high value small molecules. [ABSTRACT FROM AUTHOR]

The synthesis of the amide bond between an amine and a carboxylic acid is one of the most significant reactions in industrial pharmaceutical synthesis. Despite the apparent simplicity of synthetic methods for amide bond formation, many of these are disadvantaged by their requirement for toxic or hazardous reagents for the activation of the acid component, or poor atom economy resulting from the need for stoichiometric amounts of coupling reagents. In this context, biocatalysis has emerged as an alternative catalytic method for amide bond formation, presenting the advantages of both environmentally benign reagents and conditions and also atom economy. In this review we detail developments in the enzyme‐catalysed preparation of secondary and tertiary amides for the synthesis of pharmaceutical‐type molecules and review the applications of hydrolases, such as lipases and penicillin acylases, to these reactions. We also summarise the activity of ATP‐dependent enzymes for amide bond formation and assess their potential for the preparative synthesis of amides from carboxylic acids and amines in aqueous media. [ABSTRACT FROM AUTHOR]

Cytochrome P450 monooxygenases (P450s) play crucial roles in the cell metabolism and provide an unsurpassed diversity of catalysed reactions. Here, we report the identification and biochemical characterization of two P450s from Arthrobacter sp. a Gram-positive organism known to degrade the opium alkaloid papaverine. Combining phylogenetic and genomic analysis suggested physiological roles for P450s in metabolism and revealed potential gene clusters with redox partners facilitating the reconstitution of the P450 activities in vitro. CYP1232F1 catalyses the para demethylation of 3,4-dimethoxyphenylacetic acid to homovanillic acid while CYP1232A24 continues demethylation to 3,4-dihydroxyphenylacetic acid. Interestingly, the latter enzyme is also able to perform both demethylation steps with preference for the meta position. The crystal structure of CYP1232A24, which shares only 29% identity to previous published structures of P450s helped to rationalize the preferred demethylation specificity for the meta position and also the broader substrate specificity profile. In addition to the detailed characterization of the two P450s using their physiological redox partners, we report the construction of a highly active whole-cell Escherichia coli biocatalyst expressing CYP1232A24, which formed up to 1.77 g l−1 3,4-dihydroxyphenylacetic acid. Our results revealed the P450s' role in the metabolic pathway of papaverine enabling further investigation and application of these biocatalysts. [ABSTRACT FROM AUTHOR]

Summary: One of the major challenges in chemical synthesis is the selective oxyfunctionalization of non‐activated C‐H bonds, which can be enabled by biocatalysis using cytochrome P450 monooxygenases. In this study, we report on the characterization of the versatile CYP109Q5 from Chondromyces apiculatus DSM436, which is able to functionalize a wide range of substrates (terpenes, steroids and drugs), including the ring of β‐ionone in non‐allylic positions. The crystal structure of CYP109Q5 revealed flexibility within the active site pocket that permitted the accommodation of bulky substrates, and enabled a structure‐guided approach to engineering the enzyme. Some variants of CYP109Q5 displayed a switch in selectivity towards the non‐allylic positions of β‐ionone, allowing the simultaneous production of 2‐ and 3‐hydroxy‐β‐ionone, which are chemically challenging to synthesize and are important precursors for carotenoid synthesis. An efficient whole‐cell system finally enabled the production of up to 0.5 g l−1 hydroxylated products of β‐ionone; this system can be applied to product identification in further biotransformations. Overall, CYP109Q5 proved to be highly evolvable and active. The studies in this work demonstrate that, using rational mutagenesis, the highly versatile CYP109Q5 generalist can be progressively evolved to be an industrially valuable specialist for the synthesis of specific products. Here, we report on the identification, expression, biochemical and structural characterization as well as enzyme engineering of CYP109Q5 from the myxobacterium Chondromyces apiculatus DSM436. Overall, CYP109Q5 exhibits a versatile substrate spectra, high activities as E. coli whole‐cell biocatalyst and has shown to be highly evolvable. In addition, even non‐allylic positions of the norisoprenoid β‐ionone could be oxyfunctionalized. Thus, CYP109Q5 is highly interesting for future industrial applications. [ABSTRACT FROM AUTHOR]

The explosive xenobiotic 2,4,6-trinitrotoluene (TNT) is a major worldwide environmental pollutant and its persistence in the environment presents health and environmental concerns. The chemical structure of TNT dictates that biological detoxification pathways follow predominantly reductive transformation of the nitro groups, and as a result, TNT is notoriously recalcitrant to mineralization in the environment. Plant-based technologies to remediate this toxic pollutant rely on a solid understanding of the biochemical detoxification pathways involved. Toward this, two Arabidopsis Tau class glutathione transferases, GSTU24 and GSTU25, have been identified that catalyze the formation of three TNT-glutathionylated conjugates. These two GSTs share 79% identity yet only GSTU25 catalyzes the substitution of a nitro group for sulfur to form 2-glutathionyl-4,6-dinitrotoluene. The production of this compound is of interest because substitution of a nitro group could lead to destabilization of the aromatic ring, enabling subsequent biodegradation. To identify target amino acids within GSTU25 that might be involved in the formation of 2-glutathionyl-4,6-dinitrotoluene, the structure for GSTU25 was determined, in complex with oxidized glutathione, and used to inform site-directed mutagenesis studies. Replacement of five amino acids in GSTU24 established a conjugate profile and activity similar to that found in GSTU25. These findings contribute to the development of plant-based remediation strategies for the detoxification of TNT in the environment. [ABSTRACT FROM AUTHOR]

Traditional methods of chemical synthesis of alkaloids exhibit various problems such as lack of enantioselectivity, the use of toxic chemical and intermediates, and multiple numbers of synthetic steps. Consequently, various enzymatic methods for the formation of C−C bonds in the alkaloid skeleton have been developed. Herein, we report advances achieved in the enzymatic or chemo‐enzymatic synthesis of pharmaceutically important alkaloids that employ three C−C bond forming enzymes: two Pictet‐Spenglerases and the oxidative C−C bond forming flavoenzyme Berberine Bridge Enzyme. Protein engineering studies, improving the substrate scope of these enzymes, and thereby leading to the synthesis of non‐natural alkaloids possessing higher or newer pharmacological activities, are also discussed. Furthermore, the integration of these biocatalysts with other enzymes, in multi‐enzymatic cascades for the enantioselective synthesis of alkaloids, is also reviewed. Current results suggest that these enzymes hold great promise for the generation of C−C bonds in the selective synthesis of alkaloid compounds possessing diverse pharmacological properties. Enzymes for alkaloids: This review provides a comprehensive account of three important C−C bond forming enzymes, the Pictet‐Spenglerases Strictosidine synthase (STR) and Norcoclaurine synthase (NCS), and the oxidative C−C bond forming flavoenzyme Berberine Bridge Enzyme (BBE). Advances achieved in the enzymatic and chemo‐enzymatic synthesis of pharmaceutically important alkaloids are discussed. [ABSTRACT FROM AUTHOR]

Abstract: Amide bond formation is one of the most important reactions in pharmaceutical synthetic chemistry. The development of sustainable methods for amide bond formation, including those that are catalyzed by enzymes, is therefore of significant interest. The ATP‐dependent amide bond synthetase (ABS) enzyme McbA, from Marinactinospora thermotolerans, catalyzes the formation of amides as part of the biosynthetic pathway towards the marinacarboline secondary metabolites. The reaction proceeds via an adenylate intermediate, with both adenylation and amidation steps catalyzed within one active site. In this study, McbA was applied to the synthesis of pharmaceutical‐type amides from a range of aryl carboxylic acids with partner amines provided at 1–5 molar equivalents. The structure of McbA revealed the structural determinants of aryl acid substrate tolerance and differences in conformation associated with the two half reactions catalyzed. The catalytic performance of McbA, coupled with the structure, suggest that this and other ABS enzymes may be engineered for applications in the sustainable synthesis of pharmaceutically relevant (chiral) amides. [ABSTRACT FROM AUTHOR]

Abstract: Amide bond formation is one of the most important reactions in pharmaceutical synthetic chemistry. The development of sustainable methods for amide bond formation, including those that are catalyzed by enzymes, is therefore of significant interest. The ATP‐dependent amide bond synthetase (ABS) enzyme McbA, from Marinactinospora thermotolerans, catalyzes the formation of amides as part of the biosynthetic pathway towards the marinacarboline secondary metabolites. The reaction proceeds via an adenylate intermediate, with both adenylation and amidation steps catalyzed within one active site. In this study, McbA was applied to the synthesis of pharmaceutical‐type amides from a range of aryl carboxylic acids with partner amines provided at 1–5 molar equivalents. The structure of McbA revealed the structural determinants of aryl acid substrate tolerance and differences in conformation associated with the two half reactions catalyzed. The catalytic performance of McbA, coupled with the structure, suggest that this and other ABS enzymes may be engineered for applications in the sustainable synthesis of pharmaceutically relevant (chiral) amides. [ABSTRACT FROM AUTHOR]

We report the exploration of the evolutionary relationship between imine reductases (IREDs) and other dehydrogenases. This approach is informed by the sequence similarity between these enzyme families and the recently described promiscuous activity of IREDs for the highly reactive carbonyl compound 2,2,2-trifluoroacetophenone. Using the structure of the R-selective IRED from Streptosporangium roseum (R-IRED-Sr) as a model, β-hydroxyacid dehydrogenases (βHADs) were identified as the dehydrogenases most similar to IREDs. To understand how active site differences in IREDs and βHADs enable the reduction of predominantly C = N or C = O bonds respectively, we substituted amino acid residues in βHADs with the corresponding residues from the R-IRED-Sr and were able to increase the promiscuous activity of βHADs for C = N functions by a single amino acid substitution. Variants βHADAt_K170D and βHADAt_K170F lost mainly their keto acid reduction activity and gained the ability to catalyze the reduction of imines. Moreover, the product enantiomeric purity for a bulky imine substrate could be increased from 23% ee (R-IRED-Sr) to 97% ee (βHADAt_K170D/F_F231A) outcompeting already described IRED selectivity. [ABSTRACT FROM AUTHOR]

Abstract: Cyclic ketones bearing α‐quaternary stereocenters underwent efficient kinetic resolution using cyclohexanone monooxygenase (CHMO) from Acinetobacter calcoaceticus. Lactones possessing tetrasubstituted stereocenters were obtained with high enantioselectivity (up to >99 % ee) and complete chemoselectivity. Preparative‐scale biotransformations were exploited in conjunction with a SmI2‐mediated cyclization process to access complex, enantiomerically enriched cycloheptan‐ and cycloctan‐1,4‐diols. In a parallel approach to structurally distinct products, enantiomerically enriched ketones from the resolution with an α‐quaternary stereocenter were used in a SmI2‐mediated cyclization process to give cyclobutanol products (up to >99 % ee). [ABSTRACT FROM AUTHOR]

Abstract: Cyclic ketones bearing α‐quaternary stereocenters underwent efficient kinetic resolution using cyclohexanone monooxygenase (CHMO) from Acinetobacter calcoaceticus. Lactones possessing tetrasubstituted stereocenters were obtained with high enantioselectivity (up to >99 % ee) and complete chemoselectivity. Preparative‐scale biotransformations were exploited in conjunction with a SmI2‐mediated cyclization process to access complex, enantiomerically enriched cycloheptan‐ and cycloctan‐1,4‐diols. In a parallel approach to structurally distinct products, enantiomerically enriched ketones from the resolution with an α‐quaternary stereocenter were used in a SmI2‐mediated cyclization process to give cyclobutanol products (up to >99 % ee). [ABSTRACT FROM AUTHOR]

Biocatalytic retrosynthetic analysis of dibenz[c,e]azepines has highlighted the use of imine reductase (IRED) and ω-transaminase (ω-TA) biocatalysts to establish the key stereocentres of these molecules. Several enantiocomplementary IREDs were identified for the synthesis of ( R)- and ( S)-5-methyl-6,7-dihydro-5 H-dibenz[c,e]azepine with excellent enantioselectivity, by reduction of the parent imines. Crystallographic evidence suggests that IREDs may be able to bind one conformer of the imine substrate such that, upon reduction, the major product conformer is generated directly. ω-TA biocatalysts were also successfully employed for the production of enantiopure 1-(2-bromophenyl)ethan-1-amine, thus enabling an orthogonal route for the installation of chirality into dibenz[c,e]azepine framework. [ABSTRACT FROM AUTHOR]

Biocatalytic retrosynthetic analysis of dibenz[c,e]azepines has highlighted the use of imine reductase (IRED) and ω-transaminase (ω-TA) biocatalysts to establish the key stereocentres of these molecules. Several enantiocomplementary IREDs were identified for the synthesis of ( R)- and ( S)-5-methyl-6,7-dihydro-5 H-dibenz[c,e]azepine with excellent enantioselectivity, by reduction of the parent imines. Crystallographic evidence suggests that IREDs may be able to bind one conformer of the imine substrate such that, upon reduction, the major product conformer is generated directly. ω-TA biocatalysts were also successfully employed for the production of enantiopure 1-(2-bromophenyl)ethan-1-amine, thus enabling an orthogonal route for the installation of chirality into dibenz[c,e]azepine framework. [ABSTRACT FROM AUTHOR]

Asymmetric reductive aminations are some of the most important reactions in the preparation of active pharmaceuticals, as chiral amines feature in many of the world's most important drugs. Although many enzymes have been applied to the synthesis of chiral amines, the development of reductive amination reactions that use enzymes is attractive, as it would permit the one-step transformation of readily available prochiral ketones into chiral amines of high optical purity. However, as most natural 'reductive aminase' activities operate on keto acids, and many are able to use only ammonia as the amine donor, there is considerable scope for the engineering of natural enzymes for the reductive amination of ketones, and also for the preparation of secondary amines using alkylamines as donors. This review summarises research into the development of NAD(P)H-dependent dehydrogenases for the reductive amination of ketones, including amino acid dehydrogenases (AADHs), natural amine dehydrogenases (AmDHs), opine dehydrogenases (OpDHs) and imine reductases (IREDs). In each case knowledge of the structure and mechanism of the enzyme class is addressed, with a further description of the engineering of those enzymes for the reductive amination of ketones towards primary and also secondary amine products. [ABSTRACT FROM AUTHOR]

Glutathione transferases (GSTs) are involved in many processes in plant biochemistry, with their best characterised role being the detoxification of xenobiotics through their conjugation with glutathione. GSTs have also been implicated in noncatalytic roles, including the binding and transport of small heterocyclic ligands such as indole hormones, phytoalexins and flavonoids. Although evidence for ligand binding and transport has been obtained using gene deletions and ligand binding studies on purified GSTs, there has been no structural evidence for the binding of relevant ligands in noncatalytic sites. Here we provide evidence of noncatalytic ligand-binding sites in the phi class GST from the model plant Arabidopsis thaliana, AtGSTF2, revealed by X-ray crystallography. Complexes of the AtGSTF2 dimer were obtained with indole-3-aldehyde, camalexin, the flavonoid quercetrin and its non-rhamnosylated analogue quercetin, at resolutions of 2.00, 2.77, 2.25 and 2.38 Å respectively. Two symmetry-equivalent-binding sites ( L1) were identified at the periphery of the dimer, and one more ( L2) at the dimer interface. In the complexes, indole-3-aldehyde and quercetrin were found at both L1 and L2 sites, but camalexin was found only at the L1 sites and quercetin only at the L2 site. Ligand binding at each site appeared to be largely determined through hydrophobic interactions. The crystallographic studies support previous conclusions made on ligand binding in noncatalytic sites by AtGSTF2 based on isothermal calorimetry experiments (Dixon et al. (2011) Biochem J 438, 63-70) and suggest a mode of ligand binding in GSTs commensurate with a possible role in ligand transport. [ABSTRACT FROM AUTHOR]