A refined picture of the native amine dehydrogenase family revealed by extensive biodiversity screening.

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.

XszenFHal, a novel tryptophan 5-halogenase from Xenorhabdus szentirmaii.

Flavin-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.

Characterization of a thermotolerant ROK-type mannofructokinase from Streptococcus mitis: application to the synthesis of phosphorylated sugars.

Most of the "repressor, open reading frame, kinase" (ROK) proteins already characterized so far, and exhibiting a kinase activity, take restrictedly D-glucose as substrate. By exploring the sequenced bacterial diversity, 61 ATP-dependent kinases belonging to the ROK family have been identified and experimentally assayed for the phosphorylation of hexoses. These kinases were mainly found to be thermotolerant and highly active toward D-mannose and D-fructose with notable activities toward D-tagatose. Among them, the ATP-dependent kinase from the mesophile Streptococcus mitis (named ScrKmitis) was biochemically characterized and its substrate spectrum further studied. This enzyme possessed impressive catalytic efficiencies toward D-mannose and D-fructose of 1.5 106 s-1 M-1 and 2.7 105 s-1 M-1, respectively, but also significant ones toward D-tagatose (3.5 102 s-1 M-1) and the unnatural monosaccharides D-altrose (1.1 104 s-1 M-1) and D-talose (3.4 102 s-1 M-1). Specific activities measured for all hexoses showed a high stereopreference for D- over L-series. As proof of concept, 8 hexoses were phosphorylated in moderate to good yields, some of them described for the first time like L-sorbose-5-phosphate unusually phosphorylated in position 5. Its thermotolerance, its wide pH tolerance (from 7 to 10), and temperature range (> 85% activity between 40 and 70 °C) open the way to applications in the enzymatic synthesis of monophosphorylated hexoses.

Enzymatic Cascade Reactions for the Synthesis of Chiral Amino Alcohols from L-lysine.

Amino 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%).