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  • Further work will focus on cell

    2020-07-29

    Further work will focus on cell culture studies of three coumarin derivatives that have been found to be the most effective inhibitors on Bfd‑LDH as drug candidates. We suggest that these coumarin derivatives discovered here would provide novel lead compounds in drug design efforts to combat B. fragilis infections.
    Introduction Biocatalysis can be used to synthesize chiral building blocks, such as monomers for polymer materials, and precursors for pharmaceuticals [[1], [2], [3], [4]]. Enzymes are very suitable for catalyzing reactions with high enantioselectivity to obtain chiral products. For instance, alcohol dehydrogenases (ADHs, EC 1.1.1.X) – also known as carbonyl reductases or ketoreductases – depend on NAD(P)H to catalyze the asymmetric reduction of ketones to either (R)- or (S)-alcohols in excellent enantiomeric excess (ee) [5]. These enzymes, among others, have been applied for syntheses of pharmaceutical precursors [2,4]. Another group of redox SGC-CBP30 mg that depend on NAD(P)H are the Baeyer-Villiger monooxygenases (BVMOs) (EC 1.14.13.X). These FAD-containing enzymes can catalyze regio- and enantioselective transformations of ketones to esters or lactones, using dioxygen and NADPH. Interest in the application of BVMOs has grown, in particular for the transformation of substituted cyclic ketones to chiral lactones, for branched polyesters [[6], [7], [8], [9]]. Specifically, the recent discovery of robust BVMOs, such as the thermostable cyclohexanone monooxygenase from Thermocrispum municipale (TmCHMO), has led to great interest for exploring these monooxygenases for industrial applications, as most of the previously reported BVMOs were quite unstable [10]. Both ADHs and BVMOs rely on the cofactor NAD(P)H for catalysis, which is too expensive to apply in stoichiometric amounts, and therefore should be regenerated. There are a number of different approaches to recycle NAD(P)H [5,11,12]. One approach is to apply another enzyme which can use the oxidized NAD(P)+ and a sacrificial cheap cosubstrate to regenerate the reduced nicotinamide cofactor: the so-called “coupled-enzyme” approach. Three commonly used coenzyme regenerating enzymes are formate dehydrogenase (FDH), glucose dehydrogenase (GDH), and phosphite dehydrogenase (PTDH), all using relatively cheap substrates [12]. Instead of producing and adding these enzymes separately, the recycling enzyme can also be covalently fused to the NADPH-dependent enzyme through enzyme engineering (Scheme 1). In this way, a bifunctional and self-sufficient fusion biocatalyst is produced enabling conversion using merely one biocatalyst. Moreover, some studies on enzyme fusions provided evidence that tethering of two enzymes can improve the productivity of a multi-enzyme system [13]. In 2008 Torres Pazmiño et al. developed a platform for expressing BVMOs fused to PTDH for efficient cofactor regeneration [14,15]. Later, a few other studies reported enzyme fusions with FDH [16], GDH [17], or PTDH [[18], [19], [20]], though no study yet has compared a single biocatalyst with different regenerating enzymes.