Methylerythritol Phosphate Pathway: Enzymatic Evidence for a Rotation in the LytB/IspH‐Catalyzed Reaction

IspH/LytB, an oxygen‐sensitive [4Fe‐4S] enzyme, catalyzes the last step of the methylerythritol phosphate (MEP) pathway, a target for the development of new antimicrobial agents. This metalloenzyme converts ( E )‐4‐hydroxy‐3‐methylbut‐2‐en‐1‐yl diphosphate (HMBPP) into the two isoprenoid precursors: isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Here, the synthesis of ( S )‐[4‐ 2 H 1 ]HMBPP and ( R )‐[4‐ 2 H 1 ]HMBPP is reported together with a detailed NMR analysis of the products formed after their respective incubation with E. coli IspH/LytB in the presence of the biological reduction system used by E. coli to reduce the [4Fe‐4S] center. ( S )‐[4‐ 2 H 1 ]HMBPP was converted into [4‐ 2 H 1 ]DMAPP and ( E )‐[4‐ 2 H 1 ]IPP, whereas ( R )‐[4‐ 2 H 1 ]HMBPP yielded [4‐ 2 H 1 ]DMAPP and ( Z )‐[4‐ 2 H 1 ]IPP, hence providing the direct enzymatic evidence that the mechanism catalyzed by IspH/LytB involves a rotation of the CH 2 OH group of the substrate to display it away from the [4Fe‐4S].