D-RIBULOSE-5-PHOSPHATE 3-EPIMERASE - CLONING AND HETEROLOGOUS EXPRESSION OF THE SPINACH GENE, AND PURIFICATION AND CHARACTERIZATION OF THE RECOMBINANT ENZYME

We have achieved, to our knowledge, the first high-level heterologous expression of the gene encoding D-ribulose-5-phosphate 3-epimerase fro m any source, thereby permitting isolation and characterization of the epimerase as found in photosynthetic organisms. The extremely labile recombinant spinach (Spinacia oleracea L.) enzyme was stabilized by DL -alpha-glycerophosphate or ethanol and destabilized by D-ribulose-5-ph osphate or 2-mercaptoethanol. Despite this lability, the unprecedented ly high specific activity of the purified material indicates that the structural integrity of the enzyme is maintained throughout isolation. Ethylenediaminetetraacetate and divalent metal cations did not affect epimerase activity, thereby excluding a requirement for the latter in catalysis. As deduced from the sequence of the cloned spinach gene an d the electrophoretic mobility under denaturing conditions of the puri fied recombinant enzyme, its 25-kD subunit size was about the same as that of the corresponding epimerases of yeast and mammals. However, in contrast to these other species, the recombinant spinach enzyme was o ctameric rather than dimeric, as assessed by gel filtration and polyac rylamide gel electrophoresis under nondenaturing conditions. Western-b lot analyses with antibodies to the purified recombinant enzyme confir med that the epimerase extracted from spinach leaves is also octameric .