MOLECULAR CHARACTERIZATION OF TRANSKETOLASE (EC-2.2.1.1) ACTIVE IN THE CALVIN CYCLE OF SPINACH-CHLOROPLASTS

A cDNA encoding the Calvin cycle enzyme transketolase (TKL; EC 2.2.1.1 ) was isolated from Sorghum bicolor via subtractive differential hybri dization, and used to isolate several full-length cDNA clones for this enzyme from spinach. Functional identity of the encoded mature subuni t was shown by an 8.6-fold increase of TKL activity upon induction of Escherichia coli cells that overexpress the spinach TKL subunit under the control of the bacteriophage T-7 promoter. Chloroplast localizatio n of the cloned enzyme is shown by processing of the in vitro synthesi zed precursor upon uptake by isolated chloroplasts. Southern blot-anal ysis suggests that TKL is encoded by a single gene in the spinach geno me. TKL proteins of both higher-plant chloroplasts and the cytosol of non-photosynthetic eukaryotes are found to be unexpectedly similar to eubacterial homologues, suggesting a possible eubacterial origin of th ese nuclear genes. Chloroplast TKL is the last of the demonstrably chl oroplast-localized Calvin cycle enzymes to have been cloned and thus c ompletes the isolation of gene probes for all enzymes of the pathway i n higher plants.