Journal
CHEMISTRY & BIODIVERSITY
Volume 1, Issue 9, Pages 1367-1376Publisher
WILEY-V C H VERLAG GMBH
DOI: 10.1002/cbdv.200490099
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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-C-13(2)]-IPP stereospecifically labelled with H-2 at C(2) was prepared by incubating a D2O solution of (E)-4-hydroxy-3-methyl[1,2-C-13(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 C-13-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 H-2 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-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 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.
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