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Stereochemical Studies on the Making and Unmaking of Isopentenyl Diphosphate in Different Biological Systems
Author(s) -
Laupitz Ralf,
Gräwert Tobias,
Rieder Christoph,
Zepeck Ferdinand,
Bacher Adelbert,
Arigoni Duilio,
Rohdich Felix,
Eisenreich Wolfgang
Publication year - 2004
Publication title -
chemistry and biodiversity
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.427
H-Index - 70
eISSN - 1612-1880
pISSN - 1612-1872
DOI - 10.1002/cbdv.200490099
Subject(s) - chemistry , isomerase , stereochemistry , flavodoxin , substrate (aquarium) , enzyme , ligand (biochemistry) , nuclear magnetic resonance spectroscopy , biocatalysis , catalysis , biochemistry , reaction mechanism , receptor , ferredoxin , oceanography , geology
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 C 2 ]‐IPP stereospecifically labelled with 2 H at C(2) was prepared by incubating a D 2 O solution of ( E )‐4‐hydroxy‐3‐methyl[1,2‐ 13 C 2 ]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 13 C‐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 2 H 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 D 2 O in the course of the IspH reaction resides specifically in the H Si C(2) position of IPP and that the two isomerases mobilize specifically the same H Re C(2) ligand of their common IPP substrate. The outcome of an additional experiment, in which unlabelled IPP was incubated in D 2 O 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.