Olefin Isomerization Regiochemistries during Tandem Action of BacA and BacB on Prephenate in Bacilysin Biosynthesis

Abstract

BacA and BacB, the first two enzymes of the bacilysin pathway, convert prephenate to an exocylic regioisomer of dihydrohydroxyphenylpyruvate (ex-H(2)HPP) on the way to the epoxycyclohexanone warhead in the dipeptide antibiotic, bacilysin. BacA decarboxylates prephenate without aromatization, converting the 1,4-diene in prephenate to the endocyclic 1,3-diene in Δ(4),Δ(8)-dihydrohydroxyphenylpyruvate (en-H(2)HPP). BacB then performs an allylic isomerization to bring the diene into conjugation with the 2-ketone in the product Δ(3),Δ(5)-dihydrohydroxyphenylpyruvate (ex-H(2)HPP). To prove that BacA acts regiospecifically on one of the two prochiral olefins in prephenate, we generated 1,5,8-[(13)C]-chorismate from bacterial fermentation of 5-[(13)C]-glucose and in turn produced 2,4,6-[(13)C]-prephenate via chorismate mutase. Tandem action of BacA and BacB gave 2,4,8-[(13)C]-7R-ex-H(2)HPP, showing that BacA isomerizes only the pro-R double bond in prephenate. Nonenzymatic isomerization of the BacA product into conjugation gives only the Δ(3)E-geometric isomer of Δ(3),Δ(5)-ex-H(2)HPP. On the other hand, acceleration of the allylic isomerization by BacB gives a mixture of the E- and Z-geometric isomers of the 7R- product, indicating some rerouting of the flux, likely through dienolate geometric isomers.