Abstract
Tetracyclines are an eminent family of type II polyketides that possess a variety of decoration on the skeletons. However, apart from the oxidative modification in aureolic acid compounds, there are few cases of the further conversion of α,β-unsaturated ketones in the tetracycline D-ring. Here, we identified two reductases (TjhO5 and TjhD4), which can highly reduce the α,β-unsaturated ketone of the D-ring in 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 unusual post-modification of tetracyclines and facilitate further engineering and biocatalysis to enrich the structural diversities.
Highlights
Type II polyketides belong to a structurally diverse family of natural products with various biological activities,[1,2,3] and are closely related to the human microbiome.[4]
In path A, TjhO5 reduces the carbonyl group of C-10 to hydroxy to form the potential intermediate 22, which can be reduced by TjhO5 again to produce 16
The C-10 deoxygenation characterized in this study is inherently mechanistically different from dehydration catalyzed by KstA10 in kosinostatin biosynthesis 35, radical mechanism in apramycin, IPP and DMAPP biosynthesis[36, 37], and α-carbonyl mediated mechanism guided by the PMP-dependent enzyme SpnQ in D-forosamine biosynthesis[38] (Supplementary Fig. 16a-d)
Summary
Type II polyketides belong to a structurally diverse family of natural products with various biological activities,[1,2,3] and are closely related to the human microbiome.[4] As an essential class of type II polyketides, tetracyclines are clinically used to treat a variety of infections.[5,6] During the biosynthesis of bacterial tetracyclines, such as tetracycline (TC), chlortetracycline (CTC), oxytetracycline (OTC), and SF2575,7–10 the same intermediate 2,4-keto-anhydrotetracycline (ATC) can be further modified by various tailoring enzymes to obtain diverse tetracyclines (Fig. 1a and Supplementary Fig. 1a) [11–14]. TjhO5 along with a NAD(P)H-dependent epimerase TjhD4 accomplished the conversion of enone to alkane in D-ring of tetracyclines This process was verified to go through two different biosynthetic pathways, which involved distinct intermediates and enzymatic mechanisms based on isotope labeling experiments
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