A Flavoprotein Dioxygenase Steers Bacterial Tropone Biosynthesis via Coenzyme A-Ester Oxygenolysis and Ring Epoxidation.

Ying Duan,Jeroen S Dickschat,Nelson L Brock,Anwei Hou,Marina Toplak,Robin Teufel

doi:10.1021/jacs.1c04996

Abstract

Bacterial tropone natural products such as tropolone, tropodithietic acid, or the roseobacticides play crucial roles in various terrestrial and marine symbiotic interactions as virulence factors, antibiotics, algaecides, or quorum sensing signals. We now show that their poorly understood biosynthesis depends on a shunt product from aerobic CoA-dependent phenylacetic acid catabolism that is salvaged by the dedicated acyl-CoA dehydrogenase-like flavoenzyme TdaE. Further characterization of TdaE revealed an unanticipated complex catalysis, comprising substrate dehydrogenation, noncanonical CoA-ester oxygenolysis, and final ring epoxidation. The enzyme thereby functions as an archetypal flavoprotein dioxygenase that incorporates both oxygen atoms from O2 into the substrate, most likely involving flavin-N5-peroxide and flavin-N5-oxide species for consecutive CoA-ester cleavage and epoxidation, respectively. The subsequent spontaneous decarboxylation of the reactive enzyme product yields tropolone, which serves as a key virulence factor in rice panicle blight caused by pathogenic edaphic Burkholderia plantarii. Alternatively, the TdaE product is most likely converted to more complex sulfur-containing secondary metabolites such as tropodithietic acid from predominant marine Rhodobacteraceae (e.g., Phaeobacter inhibens).

Highlights

Bacterial natural products that feature a non-benzenoid aromatic tropone core (1, Figure 1) are of environmental and pharmaceutical importance and are produced by numerous marine and terrestrial bacteria.[1,2]. Their biosynthesis was previously linked to phenylacetic acid (2) degradation,[3,4] in which a reactive semialdehyde intermediate (3) undergoes an intramolecular condensation reaction to yield the shunt product 2-hydroxycyclohepta-1,4,6-triene-1formyl-CoA (4), which was hypothesized to be the universal tropone precursor based on its structural features (Figure 1).[5]
We show that 4 serves as a central precursor for structurally distinct bacterial tropone natural products and as a substrate for the key flavoenzyme TdaE, which is encoded by the previously reported 11-Biosynthetic gene clusters (BGCs) of marine Rhodobacteraceae and by the newly identified putative BGCs for the generation of 9 and 15 in Burkholderia spp
To identify putative BGCs of tropone natural products in pathogenic Burkholderia species, protein BLAST searches were conducted using proteins as queries that were previously associated with tropone biosynthesis in addition to enzymes involved in producing aromatic precursors de novo via the shikimate pathway.[1]

Summary

Introduction

Bacterial natural products that feature a non-benzenoid aromatic tropone core (1, Figure 1) are of environmental and pharmaceutical importance and are produced by numerous marine and terrestrial bacteria.[1,2] Their biosynthesis was previously linked to phenylacetic acid (paa) (2) degradation,[3,4] in which a reactive semialdehyde intermediate (3) undergoes an intramolecular condensation reaction to yield the shunt product 2-hydroxycyclohepta-1,4,6-triene-1formyl-CoA (4), which was hypothesized to be the universal tropone precursor based on its structural features (Figure 1).[5]. Either this aldehyde group is immediately oxidized to the more stable carboxylate (8) by the PaaZ-ALDH domain or a separate ALDH along the downstream steps of the paa catabolon that is followed by β-oxidation-like steps,[3] or a rapid spontaneous intramolecular Knoevenagel condensation to shunt product 4 occurs (Figure 1)

Results

Discussion

Conclusion