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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 P3 1 21 and was refined to 2.01 Å resolution, features two molecules (one dimer) in the asymmetric unit in complex with the redox-inactive cofactor NADPH 4 . The second form, which belongs to space group C2 1 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 P3 1 21 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., (open access.)

Native amine dehydrogenases (nat-AmDHs) have recently emerged as a potentially valuable new reservoir of enzymes for the sustainable and selective synthesis of chiral amines, catalyzing the NAD(P)H-dependent ammoniation of carbonyl compounds with high activity and selectivity. MATOUAmDH2, recently identified from the Marine Atlas of Tara Oceans Unigenes (MATOUv1) database of eukaryotic genes, displays exceptional catalytic performance against its best identified substrate, isobutyraldehyde, as well as having broader substrate scope than other nat-AmDHs. In the interests of providing a platform for the rational engineering of this and other nat-AmDHs, we have determined the structure of MATOUAmDH2 in complex with NADP + and also with the cofactor and cyclohexylamine. Monomers within the structure are representative of more open and closed conformations of the enzyme and illustrate the profound changes undergone by nat-AmDHs during the catalytic cycle. An alanine screen of active site residues revealed that M215A and L180A are more active than the wild-type enzyme for the amination of cyclohexanone with ammonia and methylamine respectively; the latter suggests that AmDHs have the potential to be engineered for the improved production of secondary amines., (© 2022 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

Chiral amine diastereomers are ubiquitous in pharmaceuticals and agrochemicals 1 , yet their preparation often relies on low-efficiency multi-step synthesis 2 . 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) collection 3 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., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Imine reductases (IREDs) have emerged as a valuable new set of biocatalysts for the asymmetric synthesis of optically active amines. The development of bioinformatics tools and searchable databases has led to the identification of a diverse range of new IRED biocatalysts that have been characterised and employed in different synthetic processes. This review describes the latest developments in the structural and mechanistic aspects of IREDs, together with synthetic applications of these enzymes, and identifies ongoing and future challenges in the field., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

NADPH-dependent oxidoreductase Q1EQE0 from Streptomyces kanamyceticus catalyzes the asymmetric reduction of the prochiral monocyclic imine 2-methyl-1-pyrroline to the chiral amine (R)-2-methylpyrrolidine with >99% ee, and is thus of interest as a potential biocatalyst for the production of optically active amines. The structures of Q1EQE0 in native form, and in complex with the nicotinamide cofactor NADPH have been solved and refined to a resolution of 2.7 Å. Q1EQE0 functions as a dimer in which the monomer consists of an N-terminal Rossman-fold motif attached to a helical C-terminal domain through a helix of 28 amino acids. The dimer is formed through reciprocal domain sharing in which the C-terminal domains are swapped, with a substrate-binding cleft formed between the N-terminal subunit of monomer A and the C-terminal subunit of monomer B. The structure is related to those of known β-hydroxyacid dehydrogenases, except that the essential lysine, which serves as an acid/base in the (de)protonation of the nascent alcohol in those enzymes, is replaced by an aspartate residue, Asp187 in Q1EQE0. Mutation of Asp187 to either asparagine or alanine resulted in an inactive enzyme., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A chimeric oxygenase, in which the P450cam domain was fused to the reductase host domains of a P450RhF from Rhodococcus sp. strain NCIMB 9784 was optimised to allow for a biotransformation at 30 mM substrate in 80% overall yield, with the linker region between P450 and FMN domain proving to be important for the effective biotransformation of (+)-camphor to 5-exo-hydroxycamphor.

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]

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]

Chiral amines feature in a large number of small molecule pharmaceuticals, and thus methods for their asymmetric synthesis are of considerable interest. Biocatalytic approaches have come to the fore in recent years as these offer advantages of superior atom economy, mild reaction conditions and excellent stereoselectivity. Advances in redox cofactor process technology have meant that oxidoreductase enzymes in particular now have growing potential as industrial catalysts for amine formation. In this review we survey recent developments in the discovery and application of oxidoreductase enzymes for amine production, including Monoamine Oxidases (MAOs), engineered and natural Amine Dehydrogenases (AmDHs), Imine Reductases (IREDs) and Reductive Aminases (RedAms), in addition to their application in enzyme cascades. [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]

Imine reductases (IREDs) are NADPH-dependent oxidoreductases that catalyse the asymmetric reduction of cyclic prochiral imines to amines, with excellent stereoselectivity. Since their discovery, stereocomplementary IREDs have been applied to the production of both ( S) and ( R) cyclic secondary amines, and the expansion in gene sequences recently identified has hinted at new substrate ranges that extend into acyclic imines and even suggest the possibility of asymmetric reductive amination from suitable ketone and amine precursors. Structural studies of various IREDs are beginning to reveal the complexities inherent in determining substrate range, stereoselectivity and mechanism in these enzymes, which represent a valuable emerging addition to the toolbox of available biocatalysts for chiral amine production. [ABSTRACT FROM AUTHOR]

Abstract: Researchers in applied biocatalysis are now reaping the rewards of intensive effort and technological developments in the sequencing of the genomes of microbial and plant species. The genomic resource contains the sequences of millions of new genes with potential application in industrial biotechnology and includes families of enzymes within discrete genomes that potentially catalyze equivalent chemical reactions. One of the key emerging characteristics of these intragenomic complements of enzymes is the impressive breadth of catalytic diversity that is observed within them. This diversity may have been acquired either in order to combat the spectrum of metabolic challenges with which the organism may be presented in its natural environment or as part of the biosynthetic machinery evolved to produce a spectrum of secondary metabolites that will prove to be advantageous in establishing a niche. Attempts have been made to functionally characterize the intragenomic complements of enzyme families catalyzing diverse reactions including carbonyl reduction, ester hydrolysis and Baeyer–Villiger oxidation, in Gram-positive bacteria, yeasts, filamentous fungi and the plant Arabidopsis thaliana. These studies are beginning to describe in detail for the first time the impressive range of catalytic potential within single organisms for attributes such as substrate range, enantioselectivity or thermostability, each of which is of interest from an enzyme discovery perspective. [ABSTRACT FROM AUTHOR]

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]