Nicotinamide is an effective non-micellar hydrotrope (solubilizer) for drugs with low aqueous solubility. To clarify the molecular basis of nicotinamide's hydrotropic effectiveness, we present here a rigorous statistical thermodynamic theory, based on the Kirkwood-Buff theory of solutions, and our recent application of it to hydrotropy. We have shown that (i) nicotinamide self-association reduces solubilization efficiency, contrary to the previous hypothesis which claimed that self-association drives solubilization and (ii) the minimum hydrotrope concentration (MHC), namely, the threshold concentration above which solubility suddenly increases, is caused not by the bulk-phase self-association of nicotinamides as has been postulated previously, but by the enhancement of nicotinamide-nicotinamide interaction around the drug molecules. We have thus established a new view of hydrotropy - it is nicotinamide's non-stoichiometric accumulation around the drug that is the basis of solubility increase above MHC.

The asymmetric dehydration of alcohols is an important process for the direct synthesis of alkenes. We report the structure and substrate specificity of the bifunctional linalool dehydratase isomerase (LinD) from the bacterium Castellaniella defragrans that catalyzes in nature the hydration of β-myrcene to linalool and the subsequent isomerization to geraniol. Enzymatic kinetic resolutions of truncated and elongated aromatic and aliphatic tertiary alcohols (C5-C15) that contain a specific signature motif demonstrate the broad substrate specificity of LinD. The three-dimensional structure of LinD from Castellaniella defragrans revealed a pentamer with active sites at the protomer interfaces. Furthermore, the structure of LinD in complex with the product geraniol provides initial mechanistic insights into this bifunctional enzyme. Site-directed mutagenesis confirmed active site amino acid residues essential for its dehydration and isomerization activity. These structural and mechanistic insights facilitate the development of hydrating catalysts, enriching the toolbox for novel bond-forming biocatalysis.

The Baeyer-Villiger monooxygenase (BVMO) 'MO14' from Rhodococcus jostii RHA1, is an enantioselective BVMO that catalyses the resolution of the model ketone substrate bicyclo[3.2.0]hept-2-en-6-one to the (1S,5R)-2-oxa lactone and the residual (1S,5R)-substrate enantiomer. This regio-plus enantioselective behaviour is highly unusual for BVMOs, which often perform enantiodivergent biotransformations of this substrate. The scaleability of the transformation was investigated using fermentor-based experiments, in which variables including gene codon optimisation, temperature and substrate concentration were investigated. E. coli cells expressing MO14 catalysed the resolution of bicyclo[3.2.0]hept-2-en-6-one to yield (1S,5R)-2-oxa lactone of >99% ee and (1S,5R)-ketone of 96% ee after 14 h at a temperature of 16 °C and a substrate concentration of 0.5 g L(-1) (4.5 mM). MO14 is thus a promising biocatalyst for the production of enantio-enriched ketones and lactones derived from the [3.2.0] platform.