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%)

Valorisation of bio-based furfurals by hybrid catalysis: towards the synthesis of new amine polymers and surfactants

International audienceThe new and highly innovative field of research called "hybrid catalysis", that combines chemo and biocatalysts, has emerged over the last two decades.[1] It is still in its infancy but already reveals numerous advantages compared to traditional processes, particularly in terms of energy savings, atom savings, but also eco-friendliness. Therefore, one of the application fields in which hybrid catalysis seems particularly attractive is that of biomass valorisation. In particular, hybrid catalysis could help achieving the production of numerous new highly valuable building blocks from complex feedstocks. Among them, 5-hydroxymethylfurfural (HMF) is described as one of the most important biobased platform molecules, with various production processes from lignocellulose. Still, its valorisation in the form of furfurylamines has been little studied, and the routes for their synthesis remain generally complex and sustainable. Nevertheless, several of these furfurylamines are very promising molecules for numerous applications, such as 5-aminomethyl-2-furancarboxilic acid (AMFC) which, thanks to its free amine and carboxylic acid moieties, can be used as a precursor of new polyamides or surfactants. Recently, several works have described the synthesis of the latter by biocatalysis, but none of them were able to directly synthesise it from HMF. Thus, we recently proposed the synthesis of AMFC directly from HMF using a hybrid one-pot/two-step process combining a platinum nanoparticle and a transaminase, both immobilised on silica beads (Fig. 1).[2] This process resulted in a 77% yield of the desired product, with 100% conversion of HMF and only one by-product, FDCA, another high value compound. A one-pot/one-step process is currently being developed using a newly discovered thermostable transaminase. In addition, this first hybrid step has since been complemented by a second step to obtain new amphiphilic molecules from AMFC, to target the production of innovative surfactants. The idea was to be able to graft aliphatic chains onto the amine function through the formation of an amide bond using sustainable catalysts and common alcohols. We therefore developed a second hybrid route, this time combining a gold nanoparticle immobilised on different supports to carry out the oxidation of the aliphatic alcohols into fatty acids, the latter then being coupled to the AMFC with the help of a CoA ligase, also thermostable. Here, 71% and 44% of amide formation could be obtained after 64h with butanol and pentanol as starting material respectively, in a one-pot/two-step process. Noteworthy, the limiting step remains the enzymatic one with 100% of alcohols to acids conversion. In addition to the overcoming of this bottleneck, a fully integrated one-pot/one-step process is also currently being developed

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

The CoA ligase from Metallosphaera sedula (MsACL) 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 MsACL 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 MsACL 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

Hybrid catalysis: towards an optimal combination of catalysts Application to HMF valorization

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

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

International audienc

CoA ligase for amide and lactam synthesis

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CoA ligase for amide and lactam synthesis

International audienc