Native amine dehydrogenases can catalyze the direct reduction of carbonyl compounds to alcohols in the absence of ammonia

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

Enzymatic cascade reactions for the synthesis of chiral amino alcohols from L-lysine

International audienceAmino alcohols are versatile compounds with a wide range of applications. For instance, they have been used as chiral scaffolds in organic synthesis. Their synthesis by conventional organic chemistry often requires tedious multi-step synthesis processes, with difficult control of the stereochemical outcome. We present a protocol to enzymatically synthetize amino alcohols starting from the readily available L-lysine in 48 h. This protocol combines two chemical reactions that are very difficult to conduct by conventional organic synthesis. In the first step, the regio- and diastereoselective oxidation of an unactivated C-H bond of the lysine side-chain is catalyzed by a dioxygenase; a second regio- and diastereoselective oxidation catalyzed by a regiodivergent dioxygenase can lead to the formation of the 1,2-diols. In the last step, the carboxylic group of the alpha amino acid is cleaved by a pyridoxal-phosphate (PLP) decarboxylase (DC). This decarboxylative step only affects the alpha carbon of the amino acid, retaining the hydroxy-substituted stereogenic center in a beta/gamma position. The resulting amino alcohols are therefore optically enriched. The protocol was successfully applied to the semipreparative-scale synthesis of four amino alcohols. Monitoring of the reactions was conducted by high performance liquid chromatography (HPLC) after derivatization by 1-fluoro-2,4-dinitrobenzene. Straightforward purification by solid-phase extraction (SPE) afforded the amino alcohols with excellent yields (93% to >95%)

Synthesis of Chiral Amines via a Bi‐Enzymatic Cascade Using an Ene‐Reductase and Amine Dehydrogenase

International audienc

One-pot bi-enzymatic cascade with an Old Yellow Enzyme and a native amine dehydrogenase to synthesize chiral amines

International audienc

Adapting an acyl CoA ligase from Metallosphaera sedula for lactam formation by structure-guided protein engineering

International audienceThe CoA ligase from Metallosphaera sedula ( Ms ACL) can be used for the chemoenzymatic synthesis of amides from carboxylic acids. In this CoA-independent conversion, the enzyme catalyzes the adenylation of a carboxylic acid with the help of ATP, followed by the uncatalyzed cleavage of acyl-AMP by a nucleophilic amine to yield an amide. With ω-amino acids as substrates this reaction may result in formation of lactams, but unfortunately the substrate preference of the wild-type enzyme is rather limited. To allow structure-based protein engineering and expand the substrate scope of the enzyme, crystal structures of Ms ACL were solved in the thioesterification conformational state with AMP, CoA and with the reaction intermediate acetyl-AMP bound in the active site. Using substrate docking and by comparing the crystals structures and sequence of Ms ACL to those of related CoA ligases, mutations were predicted which increase the affinity in the carboxylic acid binding pocket for ω-amino acids. The resulting mutations transformed a non-active enzyme into an active enzyme for ε-caprolactam synthesis, highlighting the potential of the thermophilic CoA ligase for this synthetic and biotechnologically relevant reaction

XszenFHal, a novel tryptophan 5-halogenase from Xenorhabdus szentirmaii

International audienceFlavin-dependent halogenases (FHals) catalyse the halogenation of electron-rich substrates, mainly aromatics. Halogenated compounds have many applications, as pharmaceutical, agrochemicals or as starting materials for the synthesis of complex molecules. By exploring the sequenced bacterial diversity, we discovered and characterized XszenFHal, a novel FHal from Xenorhabdus szentirmaii, a symbiotic bacterium of entomopathogenic nematode. The substrate scope of XszenFHal was examined and revealed activities towards tryptophan, indole and indole derivatives, leading to the formation of the corresponding 5-chloro products. XszenFHal makes a valuable addition to the panel of flavin-dependent halogenases already discovered and enriches the potential for biotechnology applications by allowing access to 5-halogenated indole derivatives

Synthesis of Chiral Amines via a Bi-Enzymatic Cascade Using an Ene-Reductase and Amine Dehydrogenase

Access to chiral amines with more than one stereocentre remains challenging, although an increasing number of methods are emerging. Here we developed a proof-of-concept bi-enzymatic cascade, consisting of an ene reductase and amine dehydrogenase (AmDH), to afford chiral diastereomerically enriched amines in one pot. The asymmetric reduction of unsaturated ketones and aldehydes by ene reductases from the Old Yellow Enzyme family (OYE) was adapted to reaction conditions for the reductive amination by amine dehydrogenases. By studying the substrate profiles of both reported biocatalysts, thirteen unsaturated carbonyl substrates were assayed against the best duo OYE/AmDH. Low (5 %) to high (97 %) conversion rates were obtained with enantiomeric and diastereomeric excess of up to 99 %. We expect our established bi-enzymatic cascade to allow access to chiral amines with both high enantiomeric and diastereomeric excess from varying alkene substrates depending on the combination of enzymes.Green Open Access added to TU Delft Institutional Repository ‘You share, we take care!’ – Taverne project https://www.openaccess.nl/en/you-share-we-take-care Otherwise as indicated in the copyright section: the publisher is the copyright holder of this work and the author uses the Dutch legislation to make this work public.BT/Biocatalysi

Cover Feature: Expanding the Substrate Scope of Native Amine Dehydrogenases through In Silico Structural Exploration and Targeted Protein Engineering (ChemCatChem 22/2022)

This cover feature is linked to the research article "Expanding the Substrate Scope of Native Amine Dehydrogenases through In Silico Structural Exploration and Targeted Protein Engineering" published in the same issue, deposited in HAL with the identifier cea-0378128