Stereochemical Studies on the Making and Unmaking of Isopentenyl Diphosphate in Different Biological Systems

Abstract

To investigate the unknown stereochemical course of the reaction catalyzed by the type-II isomerase, which interconverts isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), a sample of [1,2-(13)C2]-IPP stereospecifically labelled with 2H at C2 was prepared by incubating a D2O solution of (E)-4-hydroxy-3-methyl[1,2-(13)C2]but-2-enyl diphosphate with a recombinant IspH protein of Escherichia coli in the presence of NADH as a reducing agent and flavodoxin as well as flavodoxin reductase as auxiliary proteins. As monitored by 13C-NMR spectroscopy, treatment of the deuterated IPP with either type-I or type-II IPP isomerase resulted in the formation of DMAPP molecules retaining all the 2H label of the starting material. From the known stereochemical course of the type-I isomerase-catalyzed reaction, one has to conclude that the label introduced from D2O in the course of the IspH reaction resides specifically in the H(Si)-C2 position of IPP and that the two isomerases mobilize specifically the same H(Re)-C2 ligand of their common IPP substrate. The outcome of an additional experiment, in which unlabelled IPP was incubated in D2O with the type-II enzyme, demonstrates that the two isomerases also share the same preference in selecting for their reaction the (E)-methyl group of DMAPP.

Crystal Structures of Mutant IspH Proteins Reveal a Rotation of the Substrate's Hydroxymethyl Group during Catalysis

“…An additional one-electron reduction generates the anionic allyl intermediate 6 that features high basicity and therefore abstracts a proton from its surrounding. Here, proton transfer occurs exclusively from the H Si -side, 31 where the closest proton source corresponds to the hydroxyl group of PP sub .…”

New Role of Flavin as a General Acid-Base Catalyst with No Redox Function in Type 2 Isopentenyl-diphosphate Isomerase

“…During isomerization between IPP and DMAPP, proton exchange occurs selectively at C-2 and C-4 of IPP (the latter corresponds to E-methyl of DMAPP) (30,31). In addition, deprotonation and protonation at C-2 are reportedly pro-R stereospecific (31,32).…”

“…The probable interaction with Lys 8 via a water molecule might assist the protonation of FMN O-4. By this mechanism, however, it seems difficult to explain the strict specificity toward E-methyl, over Z-methyl, of DMAPP for proton exchange (30,31), the complete inactivation of type 2 IDI reconstituted with 5-deaza-FMN (7,8), and the adduct formation of substrate analogues such as eIPP with reduced FMN at its N-5 nitrogen (10,16,17).…”

IspH‐Protein von Escherichia coli – Struktur und Mechanismus

“…Die [18] Das Produktverhältnis von IPP und DMAPP ist kinetisch kontrolliert (bevorzugte Protonierung an C-3). [19] Die Abdissoziation des Produkts (IPP bzw.…”