Stereochemical Outcome at Four Stereogenic Centers during Conversion of Prephenate to Tetrahydrotyrosine by BacABGF in the Bacilysin Pathway

The first four enzymes of the bacilysin antibiotic pathway, BacABGF, convert prephenate to a tetrahydrotyrosine (H(4)Tyr) diastereomer on the way to the anticapsin warhead of the dipeptide antibiotic. BacB takes the BacA product endocyclic-Delta(4),Delta(8)-7R-dihydrohydroxyphenylpyruvate (en-H2HPP) and generates a mixture of 3E- and 3Z-olefins of the exocyclic-Delta(3),Delta(5)-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-H(4)Tyr. Incubations of the pathway enzymes in D2O yield deuterium incorporation at C-8 from Bath and then C-9 from BacB action. By H-1 NMR analysis of samples of H(4)Tyr, the stereochemistry at C-4, C-8, and C-9 can be assigned. BacG (followed by BacF) converts 3E-ex-H2HPP to 2S,4R,7R-H(4)Tyr. The 3Z isomer is instead reduced and transaminated to the opposite diastereomer at C-4, 2S,4S,7R-H(4)Tyr. Given that bacilysin has the 2S,4S stereochemistry in its anticapsin moiety, it is likely that the 2S,4S-H(4)Tyr is the diastereomer on pathway. NMR determination of the stereochemistry of the CHD samples at C-8 and C-9 allows assignment of all stereogenic centers (except C-3) in this unusual tetrahydro-aromatic amino acid building block, giving insights into and constraints on the Bath, BacB, and BacG mechanisms.