Structure and Mechanism of 3-Deoxy-d-manno-octulosonate 8-Phosphate Synthase

Abstract

3-Deoxy-D-manno-octulosonate 8-phosphate (KDO8P) synthase catalyzes the condensation of phosphoenolpyruvate (PEP) with arabinose 5-phosphate (A5P) to form KDO8P and inorganic phosphate. KDO8P is the phosphorylated precursor of 3-deoxy-D-manno-octulosonate, an essential sugar of the lipopolysaccharide of Gram-negative bacteria. The crystal structure of the Escherichia coli KDO8P synthase has been determined by multiple wavelength anomalous diffraction and the model has been refined to 2.4 Å (R-factor, 19.9%; R-free, 23.9%). KDO8P synthase is a homotetramer in which each monomer has the fold of a (␤/␣) 8 barrel. On the basis of the features of the active site, PEP and A5P are predicted to bind with their phosphate moieties 13 Å apart such that KDO8P synthesis would proceed via a linear intermediate. A reaction similar to KDO8P synthesis, the condensation of phosphoenolpyruvate, and erythrose 4-phosphate to form 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAH7P), is catalyzed by DAH7P synthase. In the active site of DAH7P synthase the two substrates PEP and erythrose 4-phosphate appear to bind in a configuration similar to that proposed for PEP and A5P in the active site of KDO8P synthase. This observation suggests that KDO8P synthase and DAH7P synthase evolved from a common ancestor and that they adopt the same catalytic strategy. 3-Deoxy-D-manno-octulosonate (KDO)1 is an 8-carbon sugar present in the lipopolysaccharide (LPS) of all Gram-negative bacteria (1). KDO provides a link between lipid A, the membrane embedded moiety of LPS, and the O-antigen, an elongated polysaccharide chain that protrudes from the bacterial outer membrane into the surrounding environment and determines the antigenic specificity of the cell. Although the composition of the O-antigen varies between species and also between strains, the inner core region containing KDO is fairly constant among all Gram-negative bacteria (2).3-Deoxy-D-manno-octulosonate 8-phosphate synthase (KDO8P synthase, EC 4.1.2.16) plays a key role in the biosynthesis of KDO. This enzyme catalyzes the aldol-type condensation of phosphoenolpyruvate (PEP) with arabinose 5-phosphate (A5P) to form KDO8P (precursor to KDO) and inorganic phosphate ( Fig. 1) (3). Dephosphorylation of KDO8P to KDO and synthesis of CMP-KDO (from CTP and KDO) occur prior to insertion of the sugar into LPS (2). Strains of Salmonella have been isolated with mutations in KDO8P synthase that confer temperature-sensitive growth (4, 5). Such strains fail to synthesize KDO at the nonpermissive temperature, which leads to the inhibition of LPS biosynthesis and, as a consequence, to the arrest of cell growth. These studies indicate that KDO8P synthase provides an essential function for bacterial homeostasis.Earlier studies have determined that the reaction of KDO8P synthesis is a sequential process in which the binding of PEP precedes the binding of A5P and the release of inorganic phosphate precedes the release of KDO8P (6). The condensation step of the reaction is stereospecific, involving the additio...

The Gene Expression and Enzyme Activity of Plant 3-Deoxy-D-Manno-2-Octulosonic Acid-8-Phosphate Synthase Are Preferentially Associated with Cell Division in a Cell Cycle-Dependent Manner

“…5). The determination of the crystal structure of E. coli Kdo-8-P synthase provided information on the threedimensional organization of the bacterial enzyme subunit, the interactions between subunits, the conformation of active sites, and the mechanism for the catalyzed reaction (Radaev et al, 2000;Wagner et al, 2000;Asojo et al, 2001). Mutations in the putative binding sites for the substrates totally inactivate (R61/T62 for binding of Ara-5-P) or greatly affect the enzyme activity (K136 for binding of PEP).…”

“…The amino acid residues shown to be essential for E. coli Kdo-8-P synthase activity (Salleh et al, 1996;Sheflyan et al, 1999;Radaev et al, 2000) are conserved in the sequence of the plant enzymes as shown in Figure 1. In the tomato Kdo-8-P synthase, residue K136 may be involved in the binding of PEP, residues R61 and T62 may be involved in the binding of Ara-5-P, and residues C164, H198, and H242 may represent structurally important residues for maintenance of active sites.…”

“…Moreover, the R61A/T62A mutation also affected the endogenous bacterial Kdo-8-P synthase activity in the wild-type strain because we observed a 10-fold decrease in the specific activity. Because the active bacterial enzyme is a homotetramer (Radaev et al, 2000), this reduction may result from the ability of the tomato Kdo- Comparison of amino acid sequences of plant Kdo-8-P synthases. Multiple alignment of the deduced amino acid sequences of Kdo-8-P synthase was realized using tomato (LeKDSA, AJ294902), pea (PsKDSA1, Y14272; and PsKDSA2, Y14273), and Arabidopsis (AtKDSA, AAD34685) sequences and the Escherichia coli Kdo-8-P synthase (EcKDSA, U18555).…”

A Single Point Mutation in 3-Deoxy-d-manno-octulosonate-8-phosphate Synthase Is Responsible for Temperature Sensitivity in a Mutant Strain of Salmonella typhimurium

“…2). Results from inhibition studies with both cyclic and acyclic product analogues (15, 16) have failed to differentiate between these mechanisms, and although no direct evidence exists, a recent structure determination favors the linear intermediate (17).In contrast to the numerous phenotypic studies on temperature-sensitive S. typhimurium mutants, and despite the prominent role of these experiments in rationalizing the study of KDO8P synthase and the wealth of knowledge concerning Escherichia coli KDO8P synthase, the nature of the S. typhimurium KDO8P synthase mutation at the enzymatic level has never been investigated. S. typhimurium AG701i50 is unable to grow at 42°C and has been used in complementation experiments to identify KDO8P synthase activity encoded in DNA libraries (18 -20).…”

Escherichia coli YrbH Is a D-Arabinose 5-Phosphate Isomerase

“…1) (12,13). The first committed step to KDO generation is catalyzed by 3-deoxy-D-manno-octulosonate 8-phosphate (KDO 8-P) synthase, which condenses D-arabinose 5-phosphate (A5P) with phosphoenolpyruvate via a stereospecific aldol-type condensation (14,15). A5P is the first intermediate unique to the KDO biosynthetic pathway, and API is the main de novo source of A5P in Gram-negative bacteria, because it is not readily available via glycolysis (16).…”