Stereochemical studies on the making and unmaking of isopentenyl diphosphate in different biological systems.
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.
Laupitz
Ralf
R
Lehrstuhl für Organische Chemie und Biochemie, Technische Universität München, Lichtenbergstrasse 4, D-85747 Garching.
Gräwert
Tobias
T
Rieder
Christoph
C
Zepeck
Ferdinand
F
Bacher
Adelbert
A
Arigoni
Duilio
D
Rohdich
Felix
F
Eisenreich
Wolfgang
W
D001412:Bacillus subtilis / Q000302:isolation & purification; D004926:Escherichia coli / Q000302:isolation & purification; D029968:Escherichia coli Proteins / Q000032:analysis / Q000737:chemistry; D045782:Hemiterpenes / Q000032:analysis* / Q000737:chemistry*; D008968:Molecular Conformation; D009943:Organophosphorus Compounds / Q000032:analysis* / Q000737:chemistry*
D029968:Escherichia coli Proteins; D045782:Hemiterpenes; D009943:Organophosphorus Compounds; C004809:isopentenyl pyrophosphate