MOESM1 of XszenFHal, a novel tryptophan 5-halogenase from Xenorhabdus szentirmaii

Additional file 1: Scheme S1. Synthesis of 5,7-dichloro-L-tryptophan (17). Table S1. Enzymes used for the reference set. Table S2. Primers used for cloning of the 148 candidate FHals and the corresponding strains used for PCR gene amplification. Table S3. Kinetic parameters of FHals for which tryptophan is the metabolic substrate; comparison with the kinetic parameters of XszenFHal. Figure S1. Matrice of the reference set. Figure S2. UHPLC traces of substrates and their corresponding chlorinated derivatives. Figure S3. MS spectra of 5-chlorotryptophan. Figure S4. Secondary metabolites from Xenorhabdus szentirmaii. Figure S5. A. UHPLC trace of the tryptophan bromination reaction by XszenFHal. B. Time course of the conversion of tryptophan by XszenFHal with NaBr over time. Figure S6. Plots for determination of kinetic parameters of XszenFHal. A. Determination of the initial velocity. B. Michaelis-Menten kinetics. Figure S7. Percent identity matrix of the sequences of the 5-tryptophan halogenases

DataSheet1_Biocatalytic Reductive Amination by Native Amine Dehydrogenases to Access Short Chiral Alkyl Amines and Amino Alcohols.PDF

Small optically active molecules, and more particularly short-chain chiral amines, are key compounds in the chemical industry and precursors of various pharmaceuticals. Their chemo-biocatalytic production on a commercial scale is already established, mainly through lipase-catalyzed resolutions leading to ChiPros™ products among others. Nevertheless, their biocatalytic synthesis remains challenging for very short-chain C4 to C5 amines due to low enantiomeric excess. To complement the possibilities recently offered by transaminases, this work describes alternative biocatalytic access using amine dehydrogenases (AmDHs). Without any protein engineering, some of the already described wild-type AmDHs (CfusAmDH, MsmeAmDH, MicroAmDH, and MATOUAmDH2) were shown to be efficient for the synthesis of hydroxylated or unfunctionalized small 2-aminoalkanes. Conversions up to 97.1% were reached at 50 mM, and moderate to high enantioselectivities were obtained, especially for (S)-1-methoxypropan-2-amine (98.1%), (S)-3-aminobutan-1-ol (99.5%), (3S)-3-aminobutan-2-ol (99.4%), and the small (S)-butan-2-amine (93.6%) with MsmeAmDH. Semi-preparative scale-up experiments were successfully performed at 150 mM substrate concentrations for the synthesis of (S)-butan-2-amine and (S)-1-methoxypropan-2-amine, the latter known as “(S)-MOIPA”. Modeling studies provided some preliminary results explaining the basis for the challenging discrimination between similarly sized substituents in the active sites of these enzymes.</p

DataSheet1_Adapting an acyl CoA ligase from Metallosphaera sedula for lactam formation by structure-guided protein engineering.pdf

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.</p

DataSheet1_Native amine dehydrogenases can catalyze the direct reduction of carbonyl compounds to alcohols in the absence of ammonia.PDF

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.</p

Achiral Hydroxypyruvaldehyde Phosphate as a Platform for Multi-Aldolases Cascade Synthesis of Diuloses and for a Quadruple Acetaldehyde Addition Catalyzed by 2‑Deoxyribose-5-Phosphate Aldolases

The 1,4-dicarbonyl unit is found in numerous molecules of biological interest, many of which are also polyhydroxylated. We developed a general, one-pot, multistep, stereoselective enzymatic method to prepare diuloses of structural diversity from achiral hydroxypyruvaldehyde phosphate (HPP) as the common electrophile for various aldolases. Surprisingly, for 2-deoxyribose-5-phosphate aldolases, HPP was an acceptor of choice for the multiple addition of acetaldehyde, widening the scope of longer-chain aldol adducts