A cDNA encoding 3-deoxy-D-manno-oct-2-ulosonate-8-phosphate synthase of Pisum sativum L. (pea) functionally complements a kdsA mutant of the Gram-negative bacterium Salmonella enterica

Recombinant plasmids encoding 3-deoxy-D-manno-oct-2-ulosonate-8-phosphate ( Kdo-8-P) synthase (KdsA; EC 4.1.2.16) were identified from a cDNA library o f Pisum sativum L. (pea) by complementing a temperature-sensitive kdsA(ts) mutant of the Gram-negative bacterium Salmonella enterica. Sequence analysi s of several inserts revealed a central open reading frame encoding a prote in of 290 amino acids with a high degree of amino acid sequence similarity to bacterial KdsA. The cDNA was confirmed by amplifying a 1,812-bp DNA frag ment from the chromosome of pea that encoded four exons around the 5'-end o f kdsA. The recombinant enzyme was subcloned, overexpressed and characteriz ed to synthesize Kdo-8-P from D-arabinose-5-phosphate and phosphoenolpyruva te. The pH optimum was 6.1 and the activity of the enzyme was neither stimu lated by the addition of divalent cations nor inhibited by EDTA. The cDNA o f kdsA could not complement Escherichia coli K-12 strain AB3257, which is d efective in all three isoenzymes (AroFGH) of 3-deoxy-D-arabino-hept-2-uloso nate-7-phosphate (Dha-7-P) synthase (EC 4.1.2.15), and neither D-erythrose- 4-phosphate nor D-ribose-5-phosphate could substitute for D-arabinose-5-pho sphate in vitro. Thus, plant cells possess a specific enzyme for the biosyn thesis of Kdo-8-P with remarkable structural and functional similarities to bacterial KdsA proteins.