Discovery of New Fe(II)/a-Ketoglutarate-Dependent Dioxygenases for Oxidation of L-Proline

Genome mining for novel Fe(II)/alpha-ketoglutarate-dependent dioxygenases (alpha KGDs) to expand the enzymatic repertoire in the oxidation of L-proline is reported. Through clustering of proteins, we predicted regio- and stereoselectivity in the hydroxylation reaction and validated this hypothesis experimentally. Two novel byproducts in the reactions with enzymes from Bacillus cereus and Streptomyces sp. were isolated, and the structures were determined to be a 3,4-epoxide and a 3,4-diol, respectively. The mechanism for the formation of the epoxide was investigated by performing an O-18-labeling experiment. We propose that the mechanism proceeds via initial cis-3-hydroxylation followed by ring closure. A biocatalytic step was run on subgram quantities of starting material without any significant optimization of the conditions. However, the substrate concentration was 40-fold higher than the usual reported titers for recombinant P450-mediated hydroxylations, showing the synthetic potential of alpha KGDs on a preparative scale.

Automated summary

Genome mining was used to discover novel Fe(II)/alpha-ketoglutarate-dependent dioxygenases for expanding the enzymatic repertoire in the oxidation of L-proline. The study successfully predicted regio- and stereoselectivity in the hydroxylation reaction, validated the hypothesis experimentally, and isolated two novel byproducts in the reactions with enzymes from Bacillus cereus and Streptomyces sp. The research also investigated the mechanism for the formation of an epoxide through O-18-labeling experiment and demonstrated the synthetic potential of alpha KGDs on a preparative scale.