Abstract

The naturally occurring diazobenzofluorenes, kinamycins, fluostatins and lomaiviticins, possess highly oxygenated A-rings, via which the last forms a dimeric pharmacophore. However, neither the A-ring transformation nor the dimerization mechanisms have been explored thus far. Here we propose a unified biosynthetic logic for the three types of antibiotics and verify one key reaction via detailed genetic and enzymatic experiments. Alp1U and Lom6 from the kinamycin and lomaiviticin biosynthesis, respectively, are shown to catalyse epoxy hydrolysis on a substrate that is obtained by chemical deacetylation of a kinamycin-pathway-derived intermediate. Thus, our study provides the first evidence for the existence of an epoxy intermediate in lomaiviticin biosynthesis. Furthermore, our results suggest that the dimerization in the lomaiviticin biosynthesis proceeds after dehydration of a product generated by Lom6.

Highlights

  • The naturally occurring diazobenzofluorenes, kinamycins, fluostatins and lomaiviticins, possess highly oxygenated A-rings, via which the last forms a dimeric pharmacophore

  • To verify our hypothesis in the kinamycin and lomaiviticin pathways, we focused on one key enzyme, the epoxy hydrolase, whose product could confirm the existence of an epoxy intermediate and an epoxidase and shed light on the dimerization precursor

  • We extended the previously proposed incomplete alp cluster[13,14] in Streptomyces ambofaciens to the far side of the genome to include region 1 and proved that an epoxy hydrolase, Alp1U, is responsible for the epoxy opening on epoxykinamycin (1) via detailed genetic and enzymatic analyses

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Summary

Introduction

The naturally occurring diazobenzofluorenes, kinamycins, fluostatins and lomaiviticins, possess highly oxygenated A-rings, via which the last forms a dimeric pharmacophore. From careful inspection of the diverse structures of the kinamycins and fluostatins, combined with our present results, we propose a unified biosynthetic logic for construction of the oxygen-substituted A-rings, and propose candidate enzymes (Fig. 1). To verify our hypothesis in the kinamycin and lomaiviticin pathways, we focused on one key enzyme, the epoxy hydrolase, whose product could confirm the existence of an epoxy intermediate and an epoxidase and shed light on the dimerization precursor. Lom[6], as the predicted counterpart of Alp1U in the lomaiviticin biosynthesis, was subsequently analysed and proved to work on 1, suggesting that a monomeric epoxy intermediate exists in the lomaiviticin biosynthesis and undergoes a similar epoxy opening process as predicted. The results presented here allow us to revise previous predictions for the lomaiviticin A-ring transformation, and facilitate future investigation of the dimerization reaction

Methods
Results
Conclusion

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