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A Facile Enzymatic Synthesis of Isotopically Labeled 2‐Methyl‐ D ‐erythritol 2,4‐Cyclodiphosphate by Spinach Chloroplast Stroma
Author(s) -
Gao Wenyun,
Raschke Maja,
Alpermann Henriette,
Zenk Meinhart H.
Publication year - 2003
Publication title -
helvetica chimica acta
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.74
H-Index - 82
eISSN - 1522-2675
pISSN - 0018-019X
DOI - 10.1002/hlca.200390301
Subject(s) - chemistry , spinach , enzyme , biochemistry , chloroplast , atp synthase , phosphate , chromoplast , plastid , gene
For the investigation of the ultimate and penultimate steps of the deoxyxylulose phosphate pathway in plants and microorganisms, and to solve intracellular transport problems, we have developed a facile enzymatic preparation of 2‐methyl‐ D ‐erythritol 2,4‐cyclodiphosphate in highly radioactive form. Use has been made of spinach chloroplast stroma, as well as the stroma of Capsicum annuum and Narcissus pseudonarcissus chromoplasts, which were shown to transform differently labeled 1‐deoxy‐ D ‐xylulose 5‐phosphate quantitatively into that cyclic diphosphate in the presence of cofactors. This method can also be exploited to synthesize milligram quantities of 13 C‐labeled cyclic diphosphate. It is shown that recombinant Escherichia coli deoxyxylulose phosphate synthase is catalytically active and can be used to synthesize the labeled starting material for further enzymatic work, as well as for feeding of intact plants. The pH and temperature stability of the deoxyxylulose 5‐phosphate and the cyclic diphosphate have been determined. Uniformly 13 C‐ or 14 C‐labeled pyruvate, and uniformly 13 C‐ or 14 C‐labeled D ‐glyceraldehyde 3‐phosphate were synthesized in almost quantitative yields from uniformly 13 C‐ or 14 C‐labeled D ‐glucose en route to deoxyxylulose 5‐phosphate.