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

Abstract

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 of Pisum sativum L. (pea) by complementing a temperature-sensitive kdsA ^ts mutant of the Gram-negative bacterium Salmonella enterica. Sequence analysis of several inserts revealed a central open reading frame encoding a protein 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 fragment from the chromosome of pea that encoded four exons around the 5′-end of kdsA. The recombinant enzyme was subcloned, overexpressed and characterized to synthesize Kdo-8-P from d-arabinose-5-phosphate and phosphoenolpyruvate. The pH optimum was 6.1 and the activity of the enzyme was neither stimulated by the addition of divalent cations nor inhibited by EDTA. The cDNA of kdsA could not complement Escherichia coli K-12 strain AB3257, which is defective in all three isoenzymes (AroFGH) of 3-deoxy-d-arabino-hept-2-ulosonate-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-phosphate in vitro. Thus, plant cells possess a specific enzyme for the biosynthesis of Kdo-8-P with remarkable structural and functional similarities to bacterial KdsA proteins.