S-Adenosyl Methionine Cofactor Modifications Enhance the Biocatalytic Repertoire of Small Molecule C-Alkylation

A tandem enzymatic strategy to enhance the scope of Calkylation of small molecules via the in situ formation of S-adenosyl methionine (SAM) cofactor analogues is described. A solventexposed channel present in the SAM-forming enzyme SalL tolerates 5'-chloro-5’-deoxyadenosine (ClDA) analogues modified at the 2position of the adenine nucleobase. Coupling SalL-catalyzed cofactor production with C-(m)ethyl transfer to coumarin substrates catalyzed by the methyltransferase (MTase) NovO forms C(m)ethylated coumarins in superior yield and greater substrate scope relative to that obtained using cofactors lacking nucleobase modifications. Establishing the molecular determinants which influence C-alkylation provides the basis to develop a late-stage enzymatic platform for the preparation of high value small molecule

Structure of the Imine Reductase from Ajellomyces dermatitidis in Three Crystal Forms

The NADPH-Dependent Imine Reductase from Ajellomyces dermatitidis (AdRedAm) catalyzes the reductive amination of certain ketones with amine donors, supplied in an equimolar ratio. We have determined the structure of AdRedAm in three forms. The first, in space group P3121, refined to 2.01 Å resolution, features two molecules (one dimer) in the asymmetric unit (asu), in complex with the redox inactive cofactor NADPH4. The second, in space group C21 and refined to 1.73 Å, has nine molecules (four and a half dimers) in the asu, each with NADP+. The third, space group P3121 and refined to 1.52 Å, had one molecule (one half-dimer) in the asu. The third structure was again in complex with NADP+ but also with the substrate 2,2-difluoroacetophenone. The different datasets permit analysis of AdRedAm in different conformational states and also reveal the molecular basis of stereoselectivity in the transformation of fluorinated acetophenone substrates by the enzyme

The Broad Aryl Acid Specificity of the Amide Bond Synthetase McbA Suggests Potential for the Biocatalytic Synthesis of Amides

Amide bond formation is one of the most important reactions in pharmaceutical synthetic chemistry. The development of sustainable methods for amide bond formation, including those that are catalyzed by enzymes, is therefore of significant interest. The ATP-dependent amide bond synthetase (ABS) enzyme McbA, from Marinactinospora thermotolerans, catalyzes the formation of amides as part of the biosynthetic pathway towards the marinacarboline secondary metabolites. The reaction proceeds via an adenylate intermediate, with both adenylation and amidation steps catalyzed within one active site. In this study, McbA was applied to the synthesis of pharmaceutical-type amides from a range of aryl carboxylic acids with partner amines provided at 1-5 molar equivalents. The structure of McbA revealed the structural determinants of aryl acid substrate tolerance and differences in conformation associated with the two half reactions catalyzed. The catalytic performance of McbA, coupled with the structure, suggest that this and other ABS enzymes may be engineered for applications in the sustainable synthesis of pharmaceutically relevant (chiral) amides

Asymmetric Synthesis of Primary and Secondary β-Fluoro-arylamines using Reductive Aminases from Fungi

The synthesis of chiral amines is of central importance to pharmaceutical chemistry, and the inclusion of fluorine atoms in drug molecules can both increase potency and slow metabolism. Optically enriched β-fluoroamines can be obtained by the kinetic resolution of racemic amines using amine transaminases (ATAs), but yields are limited to 50%, and also secondary amines are not accessible. In order to overcome these limitations, we have applied NADPH-dependent reductive aminase enzymes (RedAms) from fungal species to the reductive amination of β-fluoroacetophenones with ammonia, methylamine and allylamine as donors, to yield β-fluoro primary or secondary amines with >90% conversion and between 85 and 99% ee. In addition, the effect of the progressive introduction of fluorine atoms to the β-position of the acetophenone substrate reveals the effect of mono-, di- and tri-fluorination on the proportion of amine and alcohol in product mixtures, shedding light on the promiscuous ability of imine reductase (IRED)-type dehydrogenases to reduce fluorinated acetophenones to alcohols

The Reactivity of α-Fluoroketones with PLP Dependent Enzymes: Transaminases as Hydrodefluorinases

A chemical method for the treatment of harmful halogenated compounds that has recently become of interest is the reductive dehalogenation of carbon–halogen bonds. In the case of a fluorine atom, this process is called hydrodefluorination. While many transition metal-based approaches now exist to reductively defluorinate aromatic fluoroarenes, the cleavage of C–F bonds in aliphatic compounds is not so well-developed. Here we propose a biocatalytic approach exploiting a promiscuous activity exhibited by transaminases (TAs). Hence, a series of α-fluoroketones have been defluorinated with excellent conversions using Chromobacterium violaceum and Arthrobacter sp. TAs under mild conditions and in aqueous medium, using a stoichiometric amount of an amine (e.g. 2-propylamine) as reagent and formally releasing its oxidized form (e.g. acetone), with ammonia and hydrogen fluoride as by-products. It is also demonstrated that this process can be performed in a regio- or stereoselective fashion

Mutational Analysis of Linalool Dehydratase-Isomerase (LinD) Suggests Alcohol and Alkene Transformations are Catalyzed Using Non-Covalent Mechanisms

The interconversion of non-activated alkenes and alcohols, catalyzed by (de)hydratases, has great potential in biotechnology for the generation of fine and bulk chemicals. LinD is a cofactor-independent enzyme that catalyzes the reversible (de)hydration of the tertiary alcohol (S)-linalool to the triene beta-myrcene, and also its isomerization to the primary alcohol geraniol. Structure-informed mutagenesis of LinD, followed by activity studies, confirmed essential roles for residues C171, C180 and H129 in water activation for the hydration of beta-myrcene to linalool. However, no evidence of covalent thioterpene intermediates was found using either X-ray crystallography, mass spectrometry, or QM/MM nudged elastic band simula-tions. Labelling and NMR experiments confirmed a role for residue D39 in (de)protonation of the linalool carbon C10 in the isomerization of linalool to geraniol and also the intermediacy of beta-myrcene in this isomerization reaction. X-ray, molecular dynamics and activity studies also suggested a significant role in catalysis for a mobile methionine residue M125, which exists in substantially altered orientations in different mutant structures

Factors Controlling Contemporary Suspended Sediment Yield in the Caucasus Region

This paper discusses the joint impact of catchment complexity in topography, tectonics, climate, landuse patterns, and lithology on the suspended sediment yield (SSY, t km−2 year−1) in the Caucasus region using measurements from 244 gauging stations (GS). A Partial Least Square Regression (PLSR) was used to reveal the relationships between SSY and explanatory variables. Despite possible significant uncertainties on the SSY values, analysis of this database indicates clear spatial patterns of SSY in the Caucasus. Most catchments in the Lesser Caucasia and Ciscaucasia are characterized by relatively low SSY values (−2 year−1), the Greater Caucasus region generally have higher SSY values (more than 150–300 t km−2 year−1). Partial correlation analyses demonstrated that such proxies of topography as height above nearest drainage (HAND) and normalized steepness index (Ksn) tend to be among the most important ones. However, a PLSR analysis suggested that these variables’ influence is likely associated with peak ground acceleration (PGA). We also found a strong relationship between land cover types (e.g., barren areas and cropland) and SSY in different elevation zones. Nonetheless, adding more gauging stations into analyses and more refined characterizations of the catchments may reveal additional trends

Comparing the catalytic and structural characteristics of a ‘short’ unspecific peroxygenase (UPO) expressed in P. pastoris and E. coli

Unspecific peroxygenases (UPOs) have emerged as valuable tools for the oxygenation of non-activated carbon atoms, as they exhibit high turnovers, good stability and depend only on hydrogen peroxide as the external oxidant for activity. However, the isolation of UPOs from their natural fungal sources remains a barrier to wider application. We have cloned the gene encoding an ‘artificial’ peroxygenase (artUPO), close in sequence to the ‘short’ UPO from Marasmius rotula (MroUPO), and expressed it in both the yeast Pichia pastoris and E. coli to compare the catalytic and structural characteristics of the enzymes produced in each system. Catalytic efficiency for the UPO substrate 5-nitro-1,3-benzodioxole (NBD) was largely the same for both enzymes, and the structures also revealed few differences apart from the expected glycosylation of the yeast enzyme. However, the glycosylated enzyme displayed greater stability, as determined by nano differential scanning fluorimetry (nano-DSF) measurements. Interestingly, while artUPO hydroxylated ethylbenzene derivatives to give the (R)- alcohols, also given by a variant of the ‘long’ UPO from Agrocybe aegerita (AaeUPO), it gave the opposite (S)-series of sulfoxide products from a range of sulfide substrates, broadening the scope for application of the enzymes. The structures of artUPO reveal substantial differences to that of AaeUPO, and provide a platform for investigating the distinctive activity of this and related ’short’ UPOs

The Broad Aryl Acid Specificity of the Amide Bond Synthetase McbA Suggests Potential for the Biocatalytic Synthesis of Amides

Amide bond formation is one of the most important reactions in pharmaceutical synthetic chemistry. The development of sustainable methods for amide bond formation, including those that are catalyzed by enzymes, is therefore of significant interest. The ATP-dependent amide bond synthetase (ABS) enzyme McbA, from Marinactinospora thermotolerans, catalyzes the formation of amides as part of the biosynthetic pathway towards the marinacarboline secondary metabolites. The reaction proceeds via an adenylate intermediate, with both adenylation and amidation steps catalyzed within one active site. In this study, McbA was applied to the synthesis of pharmaceutical-type amides from a range of aryl carboxylic acids with partner amines provided at 1-5 molar equivalents. The structure of McbA revealed the structural determinants of aryl acid substrate tolerance and differences in conformation associated with the two half reactions catalyzed. The catalytic performance of McbA, coupled with the structure, suggest that this and other ABS enzymes may be engineered for applications in the sustainable synthesis of pharmaceutically relevant (chiral) amides

Multifunctional biocatalyst for conjugate reduction and reductive amination

Chiral amine diastereomers are ubiquitous in pharmaceuticals and agrochemicals,1 yet their preparation often relies on low-efficiency multi-step synthesis.2 These valuable compounds must be manufactured asymmetrically, as their biochemical properties can differ based on the chirality of the molecule. Herein, we report the discovery and characterisation of a multi-functional biocatalyst for amine synthesis, which operates using a previously unreported mechanism. This enzyme (EneIRED), identified within a metagenomic imine reductase (IRED) collection3 and originating from an unclassified Pseudomonas species, possesses an unusual active site architecture that facilitates amine-activated conjugate alkene reduction followed by reductive amination. This enzyme can couple a broad selection of α,β-unsaturated carbonyls with amines for the efficient preparation of chiral amine diastereomers baring up to three stereocentres. Mechanistic and structural studies have been carried out to delineate the order of individual steps catalysed by EneIRED which have led to a proposal for the overall catalytic cycle. This work shows that the IRED family can serve as a platform for facilitating the discovery of further enzymatic activities for application in synthetic biology and organic synthesis