Abstract
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.