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Glycosyltransferases have attracted increasing interest for the ability to construct glycosylated molecules in a facile way. However, promiscuous chemoselectivity and poor regioselectivity hinder t...

Here the synthetic utility of fluoroacetate dehalogenase RPA1163 is explored for the production of enantiomerically pure (R)-α-fluorocarboxylic acids and (R)-α-hydroxylcarboxylic acids via kinetic ...

While the mechanism of the P450-catalyzed oxidative hydroxylation of organic compounds has been studied in detail for many years, less is known about sulfoxidation. Depending upon the structure of the respective substrate, heme-Fe=O (Cpd I), heme–Fe(III)–OOH (Cpd 0), and heme–Fe(III)–H2O2 (protonated Cpd 0) have been proposed as reactive intermediates. In the present study, we consider the transformation of isosteric substrates via sulfoxidation and oxidative hydroxylation, respectively, catalyzed by regio- and enantioselective mutants of P450-BM3 which were constructed by directed evolution. 1-Thiochromanone and 1-tetralone were used as the isosteric substrates because, unlike previous studies involving fully flexible compounds such as thia-fatty acids and fatty acids, respectively, these compounds are rigid and cannot occur in a multitude of different conformations and binding modes in the large P450-BM3 binding pocket. The experimental results comprising activity and regio- and enantioselectivity, flanked by molecular dynamics computations within a time scale of 300 ns and QM/MM calculations of transition-state energies, unequivocally show that heme-Fe=O (Cpd I) is the common catalytically active intermediate in both sulfoxidation and oxidative hydroxylation.

The directed evolution of enzymes promises to eliminate the long-standing limitations of biocatalysis in organic chemistry and biotechnology—the often-observed limited substrate scope, insufficient activity, and poor regioselectivity or stereoselectivity. Saturation mutagenesis at sites lining the binding pocket with formation of focused libraries has emerged as the technique of choice, but choosing the optimal size of the randomization site and reduced amino acid alphabet for minimizing the labor-determining screening effort remains a challenge. Here, we introduce structure-guided triple-code saturation mutagenesis (TCSM) by encoding three rationally chosen amino acids as building blocks in the randomization of large multiresidue sites. In contrast to conventional NNK codon degeneracy encoding all 20 canonical amino acids and requiring the screening of more than 1015 transformants for 95% library coverage, TCSM requires only small libraries not exceeding 200–800 transformants in one library. The triple c...