Snapshots of the Catalytic Cycle of the Industrial Enzyme α‑Amino-ε-Caprolactam Racemase (ACLR) Observed Using X‑ray Crystallography

α-Amino-ε-caprolactam racemase (ACLR) is a PLP-dependent enzyme exploited in industry for the racemization of amino acid amides in dynamic kinetic resolutions that produce homochiral amino acids. We report high-resolution structures of wild-type and variant ACLRs from Rhizobium freirei, generated using the chiral substrate ACL and representative of covalent intermediates in the catalytic cycle. These complexes substantiate suggestions of a two-base mechanism, in which the PLP-binding K267 and D210 serve as proton donors in the racemization, and they constitute a robust basis on which to engineer ACLRs for improved activity as industrial biocatalysts

Broad Spectrum Enantioselective Amide Bond Synthetase from Streptoalloteichus hindustanus

The synthesis of amide bonds is one of the most frequently performed reactions in pharmaceutical synthesis, but the requirement for stoichiometric quantities of coupling agents and activated substrates in established methods has prompted interest in biocatalytic alternatives. Amide Bond Synthetases (ABSs) actively catalyze both the ATP-dependent adenylation of carboxylic acid substrates and their subsequent amidation using an amine nucleophile, both within the active site of the enzyme, enabling the use of only a small excess of the amine partner. We have assessed the ability of an ABS from Streptoalloteichus hindustanus (ShABS) to couple a range of carboxylic acid substrates and amines to form amine products. ShABS displayed superior activity to a previously studied ABS, McbA, and a remarkable complementary substrate specificity that included the enantioselective formation of a library of amides from racemic acid and amine coupling partners. The X-ray crystallographic structure of ShABS has permitted mutational mapping of the carboxylic acid and amine binding sites, revealing key roles for L207 and F246 in determining the enantioselectivity of the enzyme with respect to chiral acid and amine substrates. ShABS was applied to the synthesis of pharmaceutical amides, including ilepcimide, lazabemide, trimethobenzamide, and cinepazide, the last with 99% conversion and 95% isolated yield. These findings provide a blueprint for enabling a contemporary pharmaceutical synthesis of one of the most significant classes of small molecule drugs using biocatalysis

Engineering of a Reductive Aminase to Enable the Synthesis of a Key Intermediate to a CDK 2/4/6 Inhibitor

Biocatalytic reductive amination reactions with reductive aminases (RedAms) are emerging transformations with a high potential value for pharmaceutical synthesis. Here, we report the identification and engineering of a RedAm to catalyze a reductive amination reaction, making a key intermediate in the synthesis of an investigational cyclin-dependent kinase (CDK) inhibitor, using the relatively bulky benzylamine as a nucleophile. The engineered enzyme contains six mutations with respect to the wild-type and displays high productivity at high substrate concentrations (50-fold improved over the wild-type). After the optimized enzyme variant was identified, crystal structures of both the wild-type and mutant enzymes were solved and used to rationalize how structural changes to the RedAm improved its performance under process conditions. Results suggest that mutations affecting both substrate binding and enzyme thermostability contribute to improved enzyme performance. By enabling the multikilogram-scale synthesis of the chiral intermediate, this work highlights the versatility and industrial utility of RedAm-catalyzed reductive amination

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

Glycoprotein Labeling Using Engineered Variants of Galactose Oxidase Obtained by Directed Evolution

A directed evolution approach has been used for the generation of variants of galactose oxidase (GOase) that can selectively oxidize glycans on glycoproteins. The aldehyde function introduced on the glycans d-mannose (Man) and d-N-acetyl glucosamine (GlcNAc) by the enzyme variants could then be used to label the glycoproteins and also whole cells that display mannosides on their surface

A Covalent Succinylcysteine-like Intermediate in the Enzyme-Catalyzed Transformation of Maleate to Fumarate by Maleate Isomerase

Maleate isomerase (MI), a member of the Asp/Glu racemase superfamily, catalyzes cis−trans isomerization of the C2−C3 double bond in maleate to yield fumarate. Mutational studies, in conjunction with the structure of the C194A mutant of Nocardia farcinica MI cocrystallized with maleate, have revealed an unprecedented mode of catalysis for the superfamily in which the isomerization reaction is initiated by nucleophilic attack of cysteine at the double bond, yielding a covalent succinylcysteine-like intermediate

Engineering an Enantioselective Amine Oxidase for the Synthesis of Pharmaceutical Building Blocks and Alkaloid Natural Products

The development of cost-effective and sustainable catalytic methods for the production of enantiomerically pure chiral amines is a key challenge facing the pharmaceutical and fine chemical industries. This challenge is highlighted by the estimate that 40–45% of drug candidates contain a chiral amine, fueling a demand for broadly applicable synthetic methods that deliver target structures in high yield and enantiomeric excess. Herein we describe the development and application of a “toolbox” of monoamine oxidase variants from Aspergillus niger (MAO-N) which display remarkable substrate scope and tolerance for sterically demanding motifs, including a new variant, which exhibits high activity and enantioselectivity toward substrates containing the aminodiphenylmethane (benzhydrylamine) template. By combining rational structure-guided engineering with high-throughput screening, it has been possible to expand the substrate scope of MAO-N to accommodate amine substrates containing bulky aryl substituents. These engineered MAO-N biocatalysts have been applied in deracemization reactions for the efficient asymmetric synthesis of the generic active pharmaceutical ingredients Solifenacin and Levocetirizine as well as the natural products (<i>R</i>)-coniine, (<i>R</i>)-eleagnine, and (<i>R</i>)-leptaflorine. We also report a novel MAO-N mediated asymmetric oxidative Pictet–Spengler approach to the synthesis of (<i>R</i>)-harmicine

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

An Improved Racemase/Acylase Biotransformation for the Preparation of Enantiomerically Pure Amino Acids

Using directed evolution, a variant <i>N</i>-acetyl amino acid racemase (NAAAR G291D/F323Y) has been developed with up to 6-fold higher activity than the wild-type on a range of <i>N</i>-acetylated amino acids. The variant has been coupled with an enantiospecific acylase to give a preparative scale dynamic kinetic resolution which allows 98% conversion of <i>N</i>-acetyl-dl-allylglycine into d-allylglycine in 18 h at high substrate concentrations (50 g L^–1). This is the first example of NAAAR operating under conditions which would allow it to be successfully used on an industrial scale for the production of enantiomerically pure α-amino acids. X-ray crystal analysis of the improved NAAAR variant allowed a comparison with the wild-type enzyme. We postulate that a network of novel interactions that result from the introduction of the two side chains is the source of improved catalytic performance