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 modi cation in aureolic acid compounds, there are few cases on the further conversion of α, β-unsaturated ketones in the tetracycline D-ring. Here, we identi ed 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 CH 2 . These ndings highlight the unprecedent post-modi cation of tetracyclines and facilitate further engineering to enrich the structural diversities.

Late-stage diversification of bacterial natural products through biocatalysis

“…In a recent study, quinone oxidoreductase TjhO5 along with the NADH-dependent epimerase TjhD4 catalyze the conversion of an enone to a cycloalkane in the A-ring of unconventional tetracyclines, employing milligram-scale biocatalysis ( Supplementary Table S1 , row iv). These findings highlight an unusual post-modification of atypical tetracyclines and facilitate further engineering and biocatalysis options to enrich the structural diversities of tetracyclines ( Nie et al, 2021 ). As shown in Figure 1 , possible biocatalytic reactions of tetracycline would be reduction or alkylation of the amide group of ring A by means of oxidoreductases or transferases, respectively, oxidation of the tertiary amine of ring A, as well as halogenation of ring D by halogenating enzymes or alkylation/acylation of the phenolic OH of the ring D.…”