Molecular Cloning, Sequence Analysis and Functional Characterization of the Gene Kdsa, Encoding 3‐Deoxy‐D‐Manno‐2‐Octulosonate‐8‐Phosphate Synthase of Chlamydia Psittaci 6BC

Abstract

The kdsA gene encoding 3-deoxy-~-manno-2-octulosonate-8-phosphate (Kdo-8-P) synthase of Chlamydia psittaci 6BC was cloned by complementing the temperature-sensitive kdsA mutant Salmonella enterica serovar Typhimurium AG701i50. The sequence analysis of a recombinant DNA fragment revealed an open reading frame of 807 nucleotides which codes for a polypeptide of 269 amino acids with a high degree of similarity to known KdsA proteins. In addition, alignments of Kdo-8-P synthases with bacterial and fungal 3-deoxy-~-arabino-2-heptulosonate-7-phosphate (Dha-7-P) synthases suggested that both classes of enzymes are structurally related and may belong to a family of 2-keto-3-deoxy-aldonic acid synthases. The chlamydial protein was overexpressed and functionally characterized in vitro to synthesize Kdo-8-P from D-arabinose 5-phosphate and phosphoennlpyruvate. A chlamydial DNA region upstream of the gene exhibiting similarities to the consensus sequence of a ' " promoters of Escherichia coli was responsible for the heterologous expression of kdsA.Keywords: 3-deoxy-~-manno-2-octulosonate-8-phosphate synthase; lipopolysaccharide biosynthesis; Chlamydia.Lipopolysaccharides (LPS) are amphiphilic molecules anchored in the outer leaflet of the outer membrane of gram-negative bacteria (Rietschel et al., 1994). In general, the architecture of LPS consists of a phosphorylated and acylated p(l+6)-linked glucosainine disaccharide, termed lipid A, to which a variable carbohydrate moiety is covalently linked. For genetic and biosynthetic reasons the sugar moiety may be further divided into a lipid-A proximal core and an outer 0-antigen region (Schnaitman and Klena, 1993). As surface structures, LPS play an important role in the interaction of gram-negative bacteria with higher organisms and are responsible for many immunological and pathophysiological effects which are observed during infectious diseases. On the other hand, many cellular characteristics of gram-negative bacteria like motility or the resistance to antibiotics may be correlated with biophysical and biochemical properities of LPS (Schnaitman and Klena, 1993;Nikaido, 1994). In particular, it is known from the structural analysis of various species, as well as mutants with defects in the biosynthesis of the inner core, that 3-deoxy-D-manno-2-octu~osonic acid (Kdo) linked to lipid A is a basic constituent of all LPS and Abbreviations. Dhd-7-P, 3-deoxy-~-arabino-2-heptulosonate-7-phosphate; Kdo, 3-deoxy-~-manno-2-octulosonate ; Kdo-k-P, 3-deoxy-D-manno-2-octulosonate-8-phosphate; LPS, lipopolysaccharide.Enzymes. 3-Deoxy-~-arabino-2-heptulosonate-7-phosphate synthase (EC 4.1.2.15); 3-deoxy-~-munno-2-octulosonate-8-phosphate synthase (EC 4.1.2.1 6 ) ; CTP: 3-deoxy-D-matmo-2-octu~osonate cytidylyltransferase, CMP-Kdo synthetase (EC 2.7.7.38); 3-deoxy-~-mannu-2-octulosonate transferase (EC

A Metal Bridge between Two Enzyme Families

“…In contrast, and stemming largely from early observations on the enzyme from E. coli (16), it has been generally accepted that KDO8PS has no metal cofactor requirement. Although the identification of other bacterial (42)(43)(44)(45) and plant (46,47) KDO8P synthases has been reported, the enzymes from these organisms have yet to receive adequate characterization in order to establish their individual enzymatic properties. Therefore, a paucity of studies dealing with detailed investigations of KDO8PS from a diverse set of organisms currently exists.…”

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

“…In this respect, plant-derived Kdo-8-P synthases resembled 3-deoxy-D-arabino-hept-2-ulosonate-7-phosphate (Dha-7-P) synthases (Herrmann 1995), which initiate the shikimate pathway for the biosynthesis of a variety of aromatic compounds by the condensation of D-erythrose-4-phosphate and phosphoenolpyruvate to yield Dha-7-P and inorganic phosphate. Despite the dierent substrate speci®cities of Dha-7-P and Kdo-8-P synthases, the primary and tertiary structures of both enzymes, which have been described from bacteria and yeast, share similarities with each other (Brabetz and Brade 1997;Subramaniam et al 1998;Radaev et al 2000); however, they dier remarkably in their amino acid sequences from plant-derived Dha-7-P synthases (Dyer et al 1990;Herrmann 1995). Based on these ®ndings, the molecular characterization of Kdo-8-P synthases from plants is of interest to further understand the catalytic mechanism of these enzymes in general as well as to elucidate the biosynthesis and function of Kdo in plants.…”

Genetic and biochemical characterization of an operon involved in the biosynthesis of 3-deoxy-?-manno-octulosonic acid in Pseudomonas aeruginosa

“…At 42³C, this strain no longer synthesizes Kdo and growth ceases. In E. coli, kdsA is transcribed as part of a polycistronic mRNA from a promoter 1.2 kb upstream [18], while kdsA of Pasteurella haemolytica and Chlamydia psitacci are transcribed from promoters immediately upstream [19,20]. 1), which contains V5.0-kb of PAO1 genomic DNA and could rescue AG70li50 for growth at 42³C.…”