Hemoprotein Catalyzed Oxygenations: P450s, UPOs and Progress Towards Scalable Reactions

The selective oxygenation of non-activated carbon atoms is an ongoing synthetic challenge and biocatalysts, particularly hemoprotein oxygenases, continue to be investigated for their potential, given both their sustainable chemistry credentials and also their superior selectivity. However, issues of stability, activity and complex reaction requirements often render these biocatalytic oxygenations problematic with respect to scalable industrial processes. A continuing focus on Cyto-chromes P450 (P450s), which require a reduced nicotinamide cofactor and redox protein partners for electron transport, has now led to better catalysts and processes with a greater understanding of process requirements and limitations for both in vitro and whole-cell systems. However, the discovery and development of unspecific peroxygenases (UPOs) has also recently provided valuable complementary technology to P450-catalyzed reactions. UPOs need only hydrogen peroxide to effect ox-ygenations but are hampered by their sensitivity to peroxide and also by limited selectivity. In this perspective we survey recent developments in the engineering of proteins, cells and processes for oxygenations by these two groups of hemopro-teins and evaluate their potential and relative merits for scalable reactions

Biocatalytic Synthesis of Moclobemide Using the Amide Bond Synthetase McbA Coupled with an ATP Recycling System

The biocatalytic synthesis of amides from carboxylic acids and primary amines in aqueous media can be achieved using the ATP-dependent amide bond synthetase McbA, via an adenylate intermediate, using only 1.5 equivalents of the amine nucleophile. Following earlier studies that characterized the broad carboxylic acid specificity of McbA, we now show that, in addition to the natural amine substrate 2-phenylethylamine, a range of simple aliphatic amines, including methylamine, butylamine and hexylamine and propargylamine are coupled efficiently to the native carboxylic acid substrate 1-acetyl-9H-β-carboline-3-carboxylic acid by the enzyme, to give amide products with up to >99% conversion. The structure of wild-type McbA in its ami-dation conformation, coupled with modeling and mutational studies, reveal an amide access tunnel and a possible role for residue D201 in amide activation. Amide couplings were slower with anilines and alicyclic secondary amines such as pyrrolidine and piper-idine. The broader substrate specificity of McbA was exploited in the synthesis of the monoamine oxidase A inhibitor moclobe-mide, through the reaction of 4-chlorobenzoic acid with 1.5 equivalents of 4-(2-aminoethyl)morpholine, and utilizing polyphos-phate kinases SmPPK and AjPPK in the presence of polyphosphoric acid and 0.1 equivalents of ATP, required for recycling of the cofactor

Broadening the substrate scope of strictosidine synthases by site-directed mutagenesis

The condensation of ß-arylethylamines with carbonyl compounds (Pictet-Spengler reaction) is employed in the synthesis of tetrahydro-β-carboline and isoquinoline scaffolds which are common motifs in many naturally occurring alkaloids. These compounds exhibit a range of biological activities and are thus interesting targets for organic synthesis and medicinal chemistry. Nature’s equivalent to the Pictet-Spengler reaction is catalyzed by the so called Pictet-Spenglerases. Within this class of enzymes, strictosidine synthases (STRs, EC 4.3.3.2) have attracted attention [1-4]. These enzymes catalyse the formation of the 1,2,3,4-tetrahydro-β-carboline (S)-strictosidine, a key intermediate in the monoterpenoid indole alkaloid biosynthetic pathway in higher plants. Please click Additional Files below to see the full abstract

Structure and Mutation of Deoxypodophyllotoxin Synthase (DPS) from Podophyllum hexandrum

Deoxypodophyllotoxin synthase (DPS) is a 2-oxoglutarate (2-OG) dependent non-heme iron(II) dioxygenase that catalyzes the stereoselective ring-closing carbon-carbon bond formation of deoxypodophyllotoxin from the aryllignan (-)-yatein. Deoxypodophyllotoxin is a precursor of topoisomerase II inhibitors, which are on the World Health Organization’s list of essential medicines. Previous work has shown that DPS can accept a range of substrates, indicating it has potential in biocatalytic processes for the formation of diverse polycyclic aryllignans. Recent X-ray structures of the enzyme reveal possible roles for amino acid side chains in substrate recognition and mechanism, although a mutational analysis of DPS was not performed. Here, we present a structure of DPS at an improved resolution of 1.41 Å, in complex with the buffer molecule, Tris, coordinated to the active site iron atom. The structure has informed a mutational analysis of DPS, which suggests a role for a D224-K187 salt bridge in maintaining substrate interactions and a catalytic role for H165, perhaps as the base for the proton abstraction at the final rearomatization step. This work improves our understanding of specific residues’ contributions to the DPS mechanism and can inform future engineering of the enzyme mechanism and substrate scope for the development of a versatile biocatalyst

Recent advances in ω-transaminase-mediated biocatalysis for the enantioselective synthesis of chiral amines

Chiral amines are important components of 40-45% of small molecule pharmaceuticals and many other industrially important fine chemicals and agrochemicals. Recent advances in synthetic applications of ω-transaminases for the production of chiral amines are reviewed herein. Although a new pool of potential ω-transaminases is being continuously screened and characterized from various microbial strains, their industrial application is limited by factors such as disfavored reaction equilibrium, poor substrate scope and product inhibition. We present a closer look at recent developments in overcoming these challenges by various reaction engineering approaches. Furthermore, protein engineering techniques, which play a crucial role in improving the substrate scope of these biocatalysts and their operational stability, are also presented. Last, the incorporation of ω-transaminases in multi-enzymatic cascades, which significantly improves their synthetic applicability in the synthesis of complex chemical compounds, is detailed. This analysis of recent advances shows that ω-transaminases will continue to provide an efficient alternative to conventional catalysis for the synthesis of enantiomerically pure amines

Identification and characterization of cytochrome P450 1232A24 and 1232F1 from Arthrobacter sp. and their role in the metabolic pathway of papaverine

Cytochrome P450 monooxygenases (P450s) play crucial roles in the cell metabolism and provide an unsurpassed diversity of catalyzed reactions. Here, we report the identification and biochemical characterization of two P450s from Arthrobacter sp., a gram-positive organism known to degrade the opium alkaloid papaverine. Combining phylogenetic and genomic analysis suggested physiological roles for P450s in metabolism, and revealed potential gene clusters with redox partners facilitating the reconstitution of the P450 activities in vitro. CYP1232F1 catalyzes the para demethylation of 3,4-dimethoxyphenylacetic acid to homovanillic acid while CYP1232A24 continues demethylation to 3,4-dihydroxyphenylacetic acid. Interestingly, the latter enzyme is also able to perform both demethylation steps with preference for the meta position. The crystal structure of CYP1232A24, which shares only 29% identity to previous published structures of P450s helped to rationalize the preferred demethylation specificity for the meta position and also the broader substrate specificity profile. In addition to the detailed characterization of the two P450s using their physiological redox partners, we report the construction of a highly-active whole-cell E. coli biocatalyst expressing CYP1232A24, which formed up to 1.77 g l-1 3,4-dihydroxyphenylacetic acid. Our results revealed the P450s' role in the metabolic pathway of papaverine enabling further investigation and application of these biocatalysts

Oxidoreductase-Catalyzed Synthesis of Chiral Amines

Chiral amines are valuable constituents of many important pharmaceutical compounds and their intermediates. It is estimated that nearly 40-45% small molecule pharmaceuticals contain chiral amine scaffolds in their structures. The major challenges encountered in the chemical synthesis of enantiopure amines are the use of toxic chemicals, formation of a large number of by-products, and multi-step syntheses. To address these limitations, cost-effective biocatalytic methods are maturing and proving to be credible alternatives for the synthesis of chiral amines in enantiomerically pure forms. Herein, we report the recent progress achieved and current perspectives in the enzymatic synthesis of chiral amines using four important enzymes i.e. imine reductases, amine dehydrogenases, monoamine oxidases and cytochrome P450s. Applications to the industrial synthesis of chiral amines are highlighted. Protein engineering approaches, which play a critical role in improving or altering enzyme activity and substrate scope, are also addressed along with the discovery of pioneering enzymatic activities from nature. This survey of recent work demonstrates that enzymatic approaches to the synthesis of chiral amines will continue to be a major focus of research in biocatalytic chemistry in the years to come

Reductive Aminations by Imine Reductases: From Milligrams to Tons

The synthesis of secondary and tertiary amines through the reductive amination of carbonyl compounds is one of the most significant reactions in synthetic chemistry. Asymmetric reductive amination for the formation of chiral amines, which are required for the synthesis of pharmaceuticals and other bioactive molecules, is often achieved through transition metal catalysis, but biocatalytic methods of chiral amine production have also been a focus of interest owing to their selectivity and sustainability. The discovery of asymmetric reductive amination by imine reductase (IRED) and reductive aminase (RedAm) enzymes has served as the starting point for a new industrial approach to the production of chiral amines, leading from laboratory-scale milligram transformations to ton-scale reactions that are now described in the public domain. In this perspective we trace the development of the IRED-catalyzed reductive amination reaction from its discovery to its industrial application on kg-ton scale. In addition to surveying examples of the synthetic chemistry that has been achieved with the enzymes, the contribution of structure and protein engineering to the understanding of IRED-catalyzed reductive amination is described, and the consequent benefits for activity, selectivity and stability in the design of process suitable catalysts

Structure and Mutation of the Native Amine Dehydrogenase MATOUAmDH2

Native Amine Dehydrogenases (nat-AmDHs) have recently emerged as a potentially valuable new reservoir of enzymes for the sustainable and selective synthesis of chiral amines, catalyzing the NAD(P)H-dependent ammoniation of carbonyl compounds with high activity and selectivity. MATOUAmDH2, recently identified from the Marine Atlas of Tara Oceans Unigenes (MATOUv1) database of eukaryotic genes, displays exceptional catalytic performance against its best identified substrate, isobutyraldehyde, as well as broader substrate scope than other nat-AmDHs. In the interests of providing a platform for the rational engineering of this and other nat-AmDHs, we have determined the structure of MATOUAmDH2 in complex with NADP+ and also with the cofactor and cyclohexylamine. Monomers within the structure are representative of more open and closed conformations of the enzyme and illustrate the profound changes undergone by nat-AmDHs during the catalytic cycle. An alanine screen of active site residues revealed that M215A and L180A are more active than the wild-type enzyme for the amination of cyclohexanone with ammonia and methylamine respectively, the latter suggesting that AmDHs have the potential to be engineered for the improved production of secondary amines

Inverted binding of non-natural substrates in strictosidine synthase leads to a switch of stereochemical outcome in enzyme-catalyzed Pictet-Spengler reactions

The Pictet-Spengler reaction is a valuable route to 1,2,3,4-tetrahydro-Β-carboline (THBC) and isoquinoline scaffolds found in many important pharmaceuticals. Strictosidine synthase (STR), catalyzes the Pictet-Spengler condensation of tryptamine and the aldehyde secologanin to give (S)-strictosidine as a key intermediate in indole alkaloid biosynthesis. STRs also accept shortchain aliphatic aldehydes to give enantioenriched alkaloid products with up to 99% e.e. STRs are thus valuable asymmetric organocatalysts for applications in organic synthesis. The STR catalysis of reactions of small aldehydes gives an unexpected switch in stereopreference, leading to formation of the (R)-products. Here we report a rationale for the formation of the (R)-configured products by the STR enzyme from Ophiorrhiza pumila (OpSTR) using a combination of X-ray crystallography, mutational and molecular dynamics (MD) studies. We discovered that short chain aldehydes bind in an inverted fashion compared to secologanin leading to the inverted stereopreference for the observed (R)-product in those cases. The study demonstrates that the same catalyst can have two different productive binding modes for one substrate – but give different absolute configuration of the products by binding the aldehyde substrate differently. These results will guide future engineering of STRs and related enzymes for biocatalytic applications