Characterization of Highly Reductive Modification of Tetracycline D-Ring Reveals Enzymatic Conversion of Enone to Alkane

Abstract Tetracyclines are an eminent family of type II polyketides which possess a variety of decoration on the skeletons. However, apart from the oxidative modification in aureolic acid compounds, there are few cases on the further conversion of α, β-unsaturated ketones in the tetracycline D-ring. Here, we identified two reductases (TjhO5 and TjhD4) that highly reduced the α,β-unsaturated ketone of D-ring in the biosynthesis of unconventional tetracyclines. By identifying related intermediates and conducting isotope incorporation experiments, we demonstrated that the entire transformation could be accomplished by TjhO5 and TjhD4 collectively via two distinct pathways involving different enzymatic mechanisms. A distinctive deoxygenation mechanism was possibly involved in the TjhO5-mediated continuous reduction of C = O to CH2. These findings highlight the unprecedent post-modification of tetracyclines and facilitate further engineering to enrich the structural diversities.