Stereochemical outcome at four stereogenic centers during conversion of prephenate to tetrahydrotyrosine by BacABGF in the bacilysin pathway

Article abstract of DOI:10.1021/bi3006362

The first four enzymes of the bacilysin antibiotic pathway, BacABGF, convert prephenate to a tetrahydrotyrosine (H₄Tyr) diastereomer on the way to the anticapsin warhead of the dipeptide antibiotic. BacB takes the BacA product endocyclic-Δ⁴,Δ⁸-7R- dihydrohydroxyphenylpyruvate (en-H₂HPP) and generates a mixture of 3E- and 3Z-olefins of the exocyclic-Δ³,Δ⁵- dihydrohydroxyphenylpyruvate (ex-H₂HPP). The NADH-utilizing BacG then catalyzes a conjugate reduction, adding a pro-S hydride equivalent to C ₄ to yield tetrahydrohydroxyphenylpyruvate (H₄HPP), a transamination away (via BacF) from 2S-H₄Tyr. Incubations of the pathway enzymes in D₂O yield deuterium incorporation at C₈ from BacA and then C₉ from BacB action. By ¹H NMR analysis of samples of H₄Tyr, the stereochemistry at C₄, C₈, and C₉ can be assigned. BacG (followed by BacF) converts 3E-ex-H₂HPP to 2S,4R,7R-H₄Tyr. The 3Z isomer is instead reduced and transaminated to the opposite diastereomer at C₄, 2S,4S,7R-H₄Tyr. Given that bacilysin has the 2S,4S stereochemistry in its anticapsin moiety, it is likely that the 2S,4S-H₄Tyr is the diastereomer "on pathway". NMR determination of the stereochemistry of the CHD samples at C₈ and C₉ allows assignment of all stereogenic centers (except C₃) in this unusual tetrahydro-aromatic amino acid building block, giving insights into and constraints on the BacA, BacB, and BacG mechanisms.