The researchers initially incorporated pBoF at distinct positions in the hydrophobic cavity of the dimeric Lactococcal multidrug resistance regulatory protein, to provide distinct chemical environments where the boronic acid could accelerate the condensation reaction between racemic benzoin and hydroxylamine to the corresponding oxime. Insertion of pBoF at position 89 catalysed the reaction and discriminated between the substrate’s enantiomers, resulting in the boronic-acid-dependent oxime synthase (BOS). The group proposed that the protein environment leads to the preferential conversion of one enantiomer, allowing the kinetic resolution of the hydroxyketone product. Initial alanine scanning identified proximal residues as crucial for the catalytic activity, while further rounds of directed evolution allowed the identification of BOS_EHL, a variant containing Phe93Glu, Met8His, Ala92Leu mutations: this variant presented an E value (the ratio between catalytic rates for the two enantiomers) up to 146 and a 33-fold increase in the observed rate constant, while its reaction stopped close to 50% conversion due to the consumption of the preferred enantiomer of the benzoin substrate. After investigating the scope on the aryl ring of benzoin, the researchers collected high-resolution mass spectrometry, 11B NMR and X-ray crystallography data on BOS_EHL to understand the mechanism of action. These confirmed the boronic acid catalysis operated via the established mechanism of hydroxyl group-directed boronic acid in water. Additionally, the crystal structure revealed the hydrogen bonds between the boronic acid moiety and its neighbouring residues.
Such an interplay between the catalytic site and the protein scaffold is crucial for the new-to-nature stereoselective enzymatic reactivity demonstrated, which allows for the expansion of the biocatalytic palette with non-biological chemistries, such as this stereoselective, programmable boron biocatalysis.
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