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The Baeyer-Villiger monooxygenase (BVMO) 'MO14' from Rhodococcus jostii RHA1, is an enantioselective BVMO that catalyses the resolution of the model ketone substrate bicyclo[3.2.0]hept-2-en-6-one to the (1S,5R)-2-oxa lactone and the residual (1S,5R)-substrate enantiomer. This regio-plus enantioselective behaviour is highly unusual for BVMOs, which often perform enantiodivergent biotransformations of this substrate. The scaleability of the transformation was investigated using fermentor-based experiments, in which variables including gene codon optimisation, temperature and substrate concentration were investigated. E. coli cells expressing MO14 catalysed the resolution of bicyclo[3.2.0]hept-2-en-6-one to yield (1S,5R)-2-oxa lactone of >99% ee and (1S,5R)-ketone of 96% ee after 14 h at a temperature of 16 °C and a substrate concentration of 0.5 g L(-1) (4.5 mM). MO14 is thus a promising biocatalyst for the production of enantio-enriched ketones and lactones derived from the [3.2.0] platform.

Recombinant Escherichia coli B834 (DE3) pDB5 expressing the Rv3049c gene encoding a Baeyer-Villiger monooxygenase from Mycobacterium tuberculosis H37Rv was used for regioselective oxidations of fused bicyclic ketones. This whole-cell system represents the first recombinant Baeyer-Villiger oxidation biocatalyst that effectively resolves the racemic starting materials in this series. Within biotransformations using this organism one substrate enantiomer remains in high optical purity, while the second enantiomer is oxidized to one type of regioisomeric lactone preferably.

The enzymatic cleavage of C-C bonds in beta-diketones is, comparatively, a little studied biochemical process, but one that has important relevance to human metabolism, bioremediation and preparative biocatalysis. In recent studies, four types of enzymes have come to light that cleave C-C bonds in the beta-diketone functionality using different chemical mechanisms. OPH [oxidized poly(vinyl alcohol) hydrolase from Pseudomonas sp. strain VM15C], which cleaves nonane-4,6-dione to butyrate and pentan-2-one is a serine-triad hydrolase. Dke1 (diketone-cleaving enzyme from Acinetobacter johnsonii) is a dioxygenase, cleaving acetylacetone to methylglyoxal and acetate. Fumarylacetoacetate hydrolase cleaves fumarylacetoacetate to fumarate and acetoacetate using a water molecule, activated by a catalytic His/Asp dyad, aided by a calcium ion that both chelates the enol acid form of the substrate and indirectly positions the water for nucleophilic attack at a carbonyl group. 6-oxocamphor hydrolase cleaves nonenolizable cyclic beta-diketones and is a homologue of the crotonase superfamily, employing a catalytic His/Asp dyad to activate a water molecule for nucleophilic attack at a carbonyl group on one prochiral face of the diketone substrate, effecting desymmetrizations of symmetrical substrates.

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]

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]

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]

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]

Alcohol dehydrogenases (ADHs) are applied in industrial synthetic chemistry for the production of optically active secondary alcohols. However, the substrate spectrum of many ADHs is narrow, and few, for example, are suitable for the reduction of prochiral ketones in which the carbonyl group is bounded by two bulky and/or hydrophobic groups; so-called 'bulky-bulky' ketones. Recently two ADHs, RasADH from Ralstonia sp. DSM 6428, and SyADH from Sphingobium yanoikuyae DSM 6900, have been described, which are distinguished by their ability to accept bulky-bulky ketones as substrates. In order to examine the molecular basis of the recognition of these substrates the structures of the native and NADPH complex of RasADH, and the NADPH complex of SyADH have been determined and refined to resolutions of 1.5, 2.9 and 2.5 Å, respectively. The structures reveal hydrophobic active site tunnels near the surface of the enzymes that are well-suited to the recognition of large hydrophobic substrates, as determined by modelling of the bulky-bulky substrate n-pentyl phenyl ketone. The structures also reveal the bases for NADPH specificity and ( S)-stereoselectivity in each of the biocatalysts for n-pentyl phenyl ketone and related substrates. [ABSTRACT FROM AUTHOR]

Prochiral bicyclic diketones were transformed to a single diastereomer of 3-substituted cyclohexylamine derivatives via three consecutive biocatalytic steps. The two chiral centres were set up by a CC hydrolase (6-oxocamphor hydrolase) in the first step and by an ω-transaminase in the last step. The esterification of the intermediate keto acid was catalysed by a lipase in the second step if possible. For two substrates the CC hydrolytic step as well as the esterification could be run simultaneously in a one-pot cascade in an organic solvent. In one example, the reaction mixture of the first two steps could be directly subjected to bio-amination in an organic solvent without the need to change the reaction medium. Depending on the choice of the ω-transaminase employed and the substrate the cis- as well as the trans-diastereomers could be obtained in optically pure forms. [ABSTRACT FROM AUTHOR]

The hydrolytic cleavage of carbon–carbon bonds by β-diketone hydrolases (E.C. 3.7.1.X) is a biotransformation reaction that has received limited attention. However, studies of purified enzymes of this class have shown that they are simple hydrolases, with no complex cofactor requirement and as such, they may be a source of economical catalysts with potential application in organic synthesis. In this review, recent developments in the study of β-diketone hydrolases are surveyed, including our own work, which focuses on the first example of an asymmetric reaction catalysed by one of these enzymes—the cleavage of bicyclic β-diketones to yield keto acids of high optical purity. [Copyright &y& Elsevier]

The front cover picture, provided by Mahima Sharma and Juan Mangas ‐ Sanchez, illustrates the enzymes (AADHs, AmDHs, OpDHs and IREDs) that have been applied to date in reductive amination reactions, shown on a tree that may also bear other fruit in the form of further reductive aminase enzymes (RedAms) that are yet to be discovered or obtained through in vitro evolution. Details can be found in the review on pages 2011 – 2025 (M. Sharma, J. Mangas ‐ Sanchez, N. J. Turner, G. Grogan, Adv. Synth. Catal. 2017, 359, 2011 – 2025; DOI 10.1002/adsc.201700356). [ABSTRACT FROM AUTHOR]

The gene alr4455 from the well-studied cyanobacterium Anabaena sp. PCC 7120 encodes a crotonase orthologue that displays β-diketone hydrolase activity. Anabaena β-diketone hydrolase (ABDH), in common with 6-oxocamphor hydrolase (OCH) from Rhodococcus sp. NCIMB 9784, catalyzes the desymmetrization of bicyclo[2.2.2]octane-2,6-dione to yield [(S)-3-oxocyclohexyllacetic acid, a reaction unusual among the crotonase superfamily as the substrate is not an acyl-CoA thioester. The structure of ABDH has been determined to a resolution of 1.5 Å in both native and ligand-bound forms. ABDH forms a hexamer similar to OCH and features one active site per enzyme monomer. The arrangement of side chains in the active site indicates that while the catalytic chemistry may be conserved in OCH orthologues, the structural determinants of substrate specificity are different. In the active site of ligand-bound forms that had been cocrystallized with the bicyclic diketone substrate bicyclo[2.2.2]octane-2,6- dione was found the product of the asymmetric enzymatic retro-Claisen reaction [(S)-3-oxocyclohexyl]acetic acid. The structures of ABDH in both native and ligand-bound forms reveal further details about structural variation and modes of coenzyme A-independent activity within the crotonases and provide further evidence of a wider suprafamily of enzymes that have recruited the crotonase fold for the catalysis of reactions other than those regularly attributed to canonical superfamily members. [ABSTRACT FROM AUTHOR]