Biocatalysis: A smart and green tool for the preparation of chiral drugs.

Over the last decades, biocatalysis has achieved growing interest thanks to its potential to enable high efficiency, high yield, and eco-friendly processes aimed at the production of pharmacologically relevant compounds. Particularly, biocatalysis proved an effective and potent tool in the preparation of chiral molecules, and the recent innovations of biotechnologies and nanotechnologies open up a new era of further developments in this field. Different strategies are now available for the synthesis of chiral drugs and their intermediates. Enzymes are green tools that offer several advantages, associated both to catalysis and environmentally friendly reactants. Specifically, the use of enzymes isolated from biological sources or of whole-cell represents a valuable approach to obtain pharmaceutical products. The sustainability, the higher efficiency, and cost-effectiveness of biocatalytic reactions result in improved performance and properties that can be translated from academia to industry. In this review, we focus on biocatalytic approaches for synthesizing chiral drugs or their intermediates. Aiming to unveil the potentialities of biocatalysis systems, we discuss different examples of innovative biocatalytic approaches and their applications in the pharmaceutical industry.

The Family Keeps on Growing: Four Novel Fungal OYEs Characterized.

Aiming at expanding the portfolio of Old Yellow Enzymes (OYEs), which have been systematically studied to be employed in the chemical and pharmaceutical industries as useful biocatalysts, we decided to explore the immense reservoir of filamentous fungi. We drew from the genome of the two Ascomycetes Aspergillus niger and Botryotinia fuckeliana four new members of the OYE superfamily belonging to the classical and thermophilic-like subfamilies. The two BfOYEs show wider substrate spectra than the AnOYE homologues, which appear as more specialized biocatalysts. According to their mesophilic origins, the new enzymes neither show high thermostability nor extreme pH optimums. The crystal structures of BfOYE4 and AnOYE8 have been determined, revealing the conserved features of the thermophilic-like subclass as well as unique properties, such as a peculiar N-terminal loop involved in dimer surface interactions. For the classical representatives BfOYE1 and AnOYE2, model structures were built and analyzed, showing surprisingly wide open access to the active site cavities due to a shorter ß6-loop and a disordered capping subdomain.

A New Thermophilic Ene-Reductase from the Filamentous Anoxygenic Phototrophic Bacterium Chloroflexus aggregans.

Aiming at expanding the biocatalytic toolbox of ene-reductase enzymes, we decided to explore photosynthetic extremophile microorganisms as unique reservoir of (new) biocatalytic activities. We selected a new thermophilic ene-reductase homologue in Chloroflexus aggregans, a peculiar filamentous bacterium. We report here on the functional and structural characterization of this new enzyme, which we called CaOYE. Produced in high yields in recombinant form, it proved to be a robust biocatalyst showing high thermostability, good solvent tolerance and a wide range of pH optimum. In a preliminary screening, CaOYE displayed a restricted substrate spectrum (with generally lower activities compared to other ene-reductases); however, given the amazing metabolic ductility and versatility of Chloroflexus aggregans, further investigations could pinpoint peculiar chemical activities. X-ray crystal structure has been determined, revealing conserved features of Class III (or thermophilic-like group) of the family of Old Yellow Enzymes: in the crystal packing, the enzyme was found to assemble as dimer even if it behaves as a monomer in solution. The description of CaOYE catalytic properties and crystal structure provides new details useful for enlarging knowledge, development and application of this class of enzymes.

Developing a Library of Mannose-Based Mono- and Disaccharides: A General Chemoenzymatic Approach to Monohydroxylated Building Blocks.

Regioselective deprotection of acetylated mannose-based mono- and disaccharides differently functionalized in anomeric position was achieved by enzymatic hydrolysis. Candida rugosa lipase (CRL) and Bacillus pumilus acetyl xylan esterase (AXE) were immobilized on octyl-Sepharose and glyoxyl-agarose, respectively. The regioselectivity of the biocatalysts was affected by the sugar structure and functionalization in anomeric position. Generally, CRL was able to catalyze regioselective deprotection of acetylated monosaccharides in C6 position. When acetylated disaccharides were used as substrates, AXE exhibited a marked preference for the C2, or C6 position when C2 was involved in the glycosidic bond. By selecting the best enzyme for each substrate in terms of activity and regioselectivity, we prepared a small library of differently monohydroxylated building blocks that could be used as intermediates for the synthesis of mannosylated glycoconjugate vaccines targeting mannose receptors of antigen presenting cells.

Two new ene-reductases from photosynthetic extremophiles enlarge the panel of old yellow enzymes: CtOYE and GsOYE.

Looking for new ene-reductases with uncovered features beneficial for biotechnological applications, by mining genomes of photosynthetic extremophile organisms, we identified two new Old Yellow Enzyme homologues: CtOYE, deriving from the cyanobacterium Chroococcidiopsis thermalis, and GsOYE, from the alga Galdieria sulphuraria. Both enzymes were produced and purified with very good yields and displayed catalytic activity on a broad substrate spectrum by reducing α,ß-unsaturated ketones, aldehydes, maleimides and nitroalkenes with good to excellent stereoselectivity. Both enzymes prefer NADPH but demonstrate a good acceptance of NADH as cofactor. CtOYE and GsOYE represent robust biocatalysts showing high thermostability, a wide range of pH optimum and good co-solvent tolerance. High resolution X-ray crystal structures of both enzymes have been determined, revealing conserved features of the classical OYE subfamily as well as unique properties, such as a very long loop entering the active site or an additional C-terminal alpha helix in GsOYE. Not surprisingly, the active site of CtOYE and GsOYE structures revealed high affinity toward anions caught from the mother liquor and trapped in the anion hole where electron-withdrawing groups such as carbonyl group are engaged. Ligands (para-hydroxybenzaldehyde and 2-methyl-cyclopenten-1-one) added on purpose to study complexes of GsOYE were detected in the enzyme catalytic cavity, stacking on top of the FMN cofactor, and support the key role of conserved residues and FMN cofactor in the catalysis.