Mechanistic insight into 3-deoxy-D-manno-octulosonate-8-phosphate synthase and 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase utilizing phosphorylated monosaccharide analogues

Escherichia coli 3-deoxy-D-manno-octulosonate 8-phosphate (KDO8-P) synthase is able to utilize the five-carbon phosphorylated monosaccharide, 2-deoxyribose 5-phosphate (2dR5P), as an alternate substrate, but not D-ribose 5-phosphate (R5P) nor the four carbon analogue D-erythrose 4-phosphate (E4P). However, E. coli KDO8-P synthase in the presence of either R5P or E4P catalyzes the rapid consumption of approximately 1 mol of PEP per active site, after which consumption of PEP slows to a negligible but measurable rate. The mechanism of this abortive utilization of PEP was investigated using [2,3-(13)C(2)]-PEP and [3-F]-PEP, and the reaction products were determined by (13)C, (31)P, and (19)F NMR to be pyruvate, phosphate, and 2-phosphoglyceric acid (2-PGA). The formation of pyruvate and 2-PGA suggests that the reaction catalyzed by KDO8-P synthase may be initiated via a nucleophilic attack to PEP by a water molecule. In experiments in which the homologous enzyme, 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAH7-P) synthase was incubated with D,L-glyceraldehyde 3-phosphate (G3P) and [2,3-(13)C(2)]-PEP, pyruvate and phosphate were the predominant species formed, suggesting that the reaction catalyzed by DAH7-P synthase starts with a nucleophilic attack by water onto PEP as observed in E. coli KDO8-P synthase.     

Synthesis and evaluation of a mechanism-based inhibitor of KDO8P synthase

The enzyme 3-deoxy-D-manno-2-octulosonate-8-phosphate (KDO8P) synthase catalyzes the condensation reaction between phosphoenolpyruvate (PEP) and D-arabinose 5-phosphate (A5P) to produce KDO8P and inorganic phosphate. In attempts to investigate the lack of antibacterial activity of the most potent inhibitor of KDO8P synthase, the amino phosphonophosphate 3, we have synthesized its hydrolytically stable isosteric phosphonate analogue 4 and tested it as an inhibitor of the enzyme. The synthesis of 4 was accomplished in a one step procedure by employing the direct reductive amination in aqueous media between unprotected sugar phosphonate and glyphosate. The analogue 4 proved to be a competitive inhibitor of KDO8P synthase with respect to both substrates A5P and PEP binding. In vitro antibacterial tests against a series of different Gram-negative organisms establish that both inhibitors (3 and 4) lack antibacterial activity probably due to their reduced ability to penetrate the bacterial cell membrane.     

Function of His185 in Aquifex aeolicus 3-deoxy-D-manno-octulosonate 8-phosphate synthase

Aquifex aeolicus 3-deoxy-D-manno-octulosonate 8-phosphate synthase (KDO8PS) catalyzes the condensation of arabinose 5-phosphate (A5P) and phosphoenolpyruvate (PEP) by favoring the activation of a water molecule coordinated to the active-site metal ion. Cys11, His185, Glu222 and Asp233 are the other metal ligands. Wild-type KDO8PS is purified with Zn(2+) or Fe(2+) in the active site, but maximal activity in vitro is achieved when the endogenous metal is replaced with Cd(2+). The H185G enzyme retains 8% of the wild-type activity. ICP mass spectrometry analysis indicates that loss of His185 decreases the enzyme affinity for Fe(2+), but not for Zn(2+). However, maximal activity is again achieved by substitution of the endogenous metal with Cd(2+). We have determined the X-ray structures of the Cd(2+) H185G enzyme in its substrate-free form, and in complex with PEP, and PEP plus A5P. These structures show a normal amount of Cd(2+) bound, suggesting that coordination by His185 is not essential to retain Cd(2+) in the active site. Nonetheless, there are significant changes in the coordination sphere of Cd(2+) with respect to the wild-type enzyme, as the carboxylate moiety of PEP binds directly to the metal ion and replaces water and His185 as ligands. These observations indicate that the primary function of His185 in A.aeolicus KDO8PS is to orient PEP in the active site of the enzyme in such a way that a water molecule on the sinister (si) side of PEP can be activated by direct coordination to the metal ion.     

The catalytic and conformational cycle of Aquifex aeolicus KDO8P synthase: role of the L7 loop

KDO8P synthase catalyzes the condensation of arabinose 5-phosphate (A5P) and phosphoenolpyruvate (PEP) to form the 8-carbon sugar KDO8P and inorganic phosphate (P(i)). The X-ray structure of the wild-type enzyme shows that when both PEP and A5P bind, the active site becomes isolated from the environment due to a conformational change of the L7 loop. The structures of the R106G mutant, without substrates, and with PEP and PEP plus A5P bound, were determined and reveal that in R106G closure of the L7 loop is impaired. The structural perturbations originating from the loss of the Arg(106) side chain point to a role of the L2 loop in stabilizing the closed conformation of the L7 loop. Despite the increased exposure of the R106G active site, no abnormal reaction of PEP with water was observed, ruling out the hypothesis that the primary function of the L7 loop is to shield the active site from bulk solvent during the condensation reaction. However, the R106G enzyme displays several kinetic abnormalities on both the substrate side (smaller K(m)(PEP), larger K(i)(A5P) and K(m)(A5P)) and the product side (smaller K(i)(Pi) and K(i)(KDO8P)) of the reaction. As a consequence, the mutant enzyme is less severely inhibited by A5P and more severely inhibited by P(i) and KDO8P. Simulations of the flux of KDO8P synthesis under metabolic steady-state conditions (constant concentration of reactants and products over time) suggest that in vivo R106G is expected to perform optimally in a narrower range of substrate and product concentrations than the wild-type enzyme.     

Structure and mechanism of 3-deoxy-D-manno-octulosonate 8-phosphate synthase

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 A (R-factor, 19.9%; R-free, 23.9%). KDO8P synthase is a homotetramer in which each monomer has the fold of a (beta/alpha)(8) barrel. On the basis of the features of the active site, PEP and A5P are predicted to bind with their phosphate moieties 13 A 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.     

Structural and mechanistic investigation of 3-deoxy-D-manno-octulosonate-8-phosphate synthase by solid-state REDOR NMR

15N?(31)P? REDOR NMR experiments were applied to lyophilized binary complexes of 3-deoxy-D-manno-2-octulosonate-8-phosphate synthase (KDO8PS), with each of its natural substrates, phosphoenolpyruvate (PEP) and arabinose-5-phsophate (A5P), and with a mechanism-based inhibitor (K(i) = 0.4 microM), directly characterizing the active site basic residues involved in the binding of their carboxylate and phosphate moieties. KDO8PS was labeled uniformly with (15)N or [eta-(15)N(2)]Arg, and the ligands were selectively labeled with (13)C and (15)N. The NMR data established that PEP is bound by KDO8PS via a preserved set of structurally rigid and chemically unique Arg and Lys residues, with 5 A (upper limit) between epsilon-(15)N of this Lys and (31)P of PEP. A5P is bound in its cyclic forms to KDO8PS via a different set of Lys and Arg residues. The two sets arise from adjacent subsites that are capable of independent and sufficiently strong binding. The inhibitor is best characterized as an A5P-based substrate analogue inhibitor of KDO8PS. Five mutants in which highly conserved arginines were replaced with alanines were prepared and kinetically characterized. Our solid-state NMR observations complement the crystallographic structure of KDO8PS, and in combination with the mutagenesis results enable tentative assignment of the NMR-identified active site residues. Lys-138 and Arg-168 located at the most recessed part of the active site cavity are the chemically distinct and structurally rigid residues that bind PEP phosphate; R168A resulted in 0.1% of wild-type activity. Arg-63, exposed at the opening of the active site barrel, is the flexible residue with a generic chemical shift that binds A5P; R63A resulted in complete deactivation. The mechanistic implications of our results are discussed.     

Substrate and metal complexes of 3-deoxy-D-manno-octulosonate-8-phosphate synthase from Aquifex aeolicus at 1.9-A resolution. Implications for the condensation mechanism

3-Deoxy-D-manno-octulosonate-8-phosphate synthase (KDO8PS) from the hyperthermophilic bacterium Aquifex aeolicus differs from its Escherichia coli counterpart in the requirement of a divalent metal for activity (Duewel, H. S., and Woodard, R. W. (2000) J. Biol. Chem. 275, 22824-22831). Here we report the crystal structure of the A. aeolicus enzyme, which was determined by molecular replacement using E. coli KDO8PS as a model. The structures of the metal-free and Cd(2+) forms of the enzyme were determined in the uncomplexed state and in complex with various combinations of phosphoenolpyruvate (PEP), arabinose 5-phosphate (A5P), and erythrose 4-phosphate (E4P). Like the E. coli enzyme, A. aeolicus KDO8PS is a homotetramer containing four distinct active sites at the interface between subunits. The active site cavity is open in the substrate-free enzyme or when either A5P alone or PEP alone binds, and becomes isolated from the aqueous phase when both PEP and A5P (or E4P) bind together. In the presence of metal, the enzyme is asymmetric and appears to alternate catalysis between the active sites located on one face of the tetramer and those located on the other face. In the absence of metal, the asymmetry is lost. Details of the active site that may be important for catalysis are visible at the high resolution achieved in these structures. Most notably, the shape of the PEP-binding pocket forces PEP to assume a distorted geometry at C-2, which might anticipate the conversion from sp(2) to sp(3) hybridization occurring during intermediate formation and which may modulate PEP reactivity toward A5P. Two water molecules are located in van der Waals contact with the si and re sides of C-2(PEP), respectively. Abstraction of a proton from either of these water molecules by a protein group is expected to elicit a nucleophilic attack of the resulting hydroxide ion on the nearby C-2(PEP), thus triggering the beginning of the catalytic cycle.     

Crystal structures of Escherichia coli KDO8P synthase complexes reveal the source of catalytic irreversibility

The enzyme 3-deoxy-D-manno-2-octulosonate-8-phosphate synthase (KDO8PS) catalyses the condensation of arabinose 5-phosphate (A5P) and phosphoenol pyruvate (PEP) to obtain 3-deoxy-D-manno-2-octulosonate-8-phosphate (KDO8P). We have elucidated initial modes of ligand binding in KDO8PS binary complexes by X-ray crystallography. Structures of the apo-enzyme and of binary complexes with the substrate PEP, the product KDO8P and the catalytically inactive 1-deoxy analog of arabinose 5-phosphate (1dA5P) were obtained. The KDO8PS active site resembles an irregular funnel with positive electrostatic potential situated at the bottom of the PEP-binding sub-site, which is the primary attractive force towards negatively charged phosphate moieties of all ligands. The structures of the ligand-free apo-KDO8PS and the binary complex with the product KDO8P visualize for the first time the role of His202 as an active-site gate. Examination of the crystal structures of KDO8PS with the KDO8P or 1dA5P shows these ligands bound to the enzyme in the PEP-binding sub-site, and not as expected to the A5P sub-site. Taken together, the structures presented here strengthen earlier evidence that this enzyme functions predominantly through positional catalysis, map out the roles of active-site residues and provide evidence that explains the total lack of catalytic reversibility.     

The use of (E)- and (Z)-phosphoenol-3-fluoropyruvate as mechanistic probes reveals significant differences between the active sites of KDO8P and DAHP synthases

The enzymes 3-deoxy-d-manno-2-octulosonate-8-phosphate (KDO8P) synthase and 3-deoxy-d-arabino-2-heptulosonate-7-phosphate (DAHP) synthase catalyze a similar aldol-type condensation between phosphoenolpyruvate (PEP) and the corresponding aldose: arabinose 5-phosphate (A5P) and erythrose 4-phosphate (E4P), respectively. While KDO8P synthase is metal-dependent in one class of organisms and metal-independent in another, only a metal-dependent class of DAHP synthases has thus far been identified in nature. We have used catalytically active E and Z isomers of phosphoenol-3-fluoropyruvate [(E)- and (Z)-FPEP, respectively] as mechanistic probes to characterize the differences and/or the similarities between the metal-dependent and metal-independent KDO8P synthases as well as between the metal-dependent KDO8P synthase and DAHP synthase. The direct evidence of the overall stereochemistry of the metal-dependent Aquifex pyrophilus KDO8P synthase (ApKDO8PS) reaction was obtained by using (E)- and (Z)-FPEPs as alternative substrates and by subsequent (19)F NMR analysis of the products. The results reveal the si face addition of the PEP to the re face of the carbonyl of A5P, and establish that the stereochemistry of ApKDO8PS is identical to that of the metal-independent Escherichia coli KDO8P synthase enzyme (EcKDO8PS). In addition, both ApKDO8PS and EcKDO8PS enzymes exhibit high selectivity for (E)-FPEP versus (Z)-FPEP, the relative k(cat)/K(m) ratios being 100 and 33, respectively. In contrast, DAHP synthase does not discriminate between (E)- and (Z)-FPEP (the k(cat)/K(m) being approximately 7 x 10(-)(3) microM(-)(1) s(-)(1) for both compounds). The pre-steady-state burst experiments for EcKDO8PS showed that product release is rate-limiting for the reactions performed with either PEP, (E)-FPEP, or (Z)-FPEP, although the rate constants, for both product formation and product release, were lower for the fluorinated analogues than for PEP [125 and 2.3 s(-)(1) for PEP, 2.5 and 0.2 s(-)(1) for (E)-FPEP, and 9 and 0.1 s(-)(1) for (Z)-FPEP, respectively]. The observed data indicate substantial differences in the PEP subsites and open the opportunity for the design of selective inhibitors against these two families of enzymes.     

Synthesis and evaluation of tetrahedral intermediate mimic inhibitors of 3-deoxy-d-manno-octulosonate 8-phosphate synthase

3-Deoxy-d-manno-octulosonate 8-phosphate (KDO8P) synthase catalyses the first committed step in the biosynthesis of 3-deoxy-d-manno-octulosonate (KDO), an important component of the lipopolysaccharide of Gram-negative bacteria. The pathway for KDO biosynthesis has been identified as a potential target of antibacterial drug design. The reaction catalysed by KDO8P synthase is an aldol-like condensation between phosphoenolpyruvate (PEP) and d-arabinose 5-phosphate (A5P) and proceeds through a bisphosphorylated tetrahedral intermediate. In this study a bisphosphate analogue of the tetrahedral intermediate was synthesised and was found to inhibit the metal-dependent KDO8P synthase from Neisseriameningitidis and the metal-dependent KDO8P synthase from Acidithiobacillus ferrooxidans with inhibition constants in the low micromolar range. Additionally, monophosphorylated inhibitors were synthesised to determine the relative importance of the two phosphate groups of this bisphosphate analogue for enzyme inhibition. The removal of either of these two phosphate groups gave less potent inhibitors for both enzymes.     

Synthesis and antibacterial activity of mechanism-based inhibitors of KDO8P synthase and DAH7P synthase

KDO8PS (3-deoxy-D-manno-2-octulosonate-8-phosphate synthase) and DAH7PS (3-deoxy-D-arabino-2-heptulosonate-7-phosphate synthase) are attractive targets for the development of new anti-infectious agents. Both enzymes appear to proceed via a common mechanism involving the reaction of phosphoenolpyruvate (PEP) with arabinose 5-phosphate or erythrose-4-phosphate, to produce the corresponding ulosonic acids, KDO8P and DAH7P, respectively. The synthesis of new inhibitors closely related to the supposed tetrahedral intermediate substrates for the enzymes is described. The examination of the antibacterial activity of these derivatives is reported.     

Common basis for the mechanism of metallo and non-metallo KDO8P synthases

The three-dimensional structures of metal and non-metal enzymes that catalyze the same reaction are often quite different, a clear indication of convergent evolution. However, there are interesting cases in which the same scaffold supports both a metal and a non-metal catalyzed reaction. One of these is 3-deoxy-D-manno-octulosonate 8-phosphate (KDO8P) synthase (KDO8PS), a bacterial enzyme that catalyzes the synthesis of KDO8P and inorganic phosphate (P_i) from phosphoenolpyruvate (PEP), arabinose 5-phosphate (A5P), and water. This reaction is one of the key steps in the biosynthesis of bacterial endotoxins. The evolutionary tree of KDO8PS is evenly divided between metal and non-metal forms, both having essentially identical structures. Mutagenesis and crystallographic studies suggest that one or two residues at most determine whether or not KDO8PS requires a metal for function, a clear example of “minimalist evolution”. Quantum mechanical/molecular mechanical (QM/MM) simulations of both the enzymatic and non-enzymatic synthesis of KDO8P have revealed the mechanism underlying the switch between metal and non-metal dependent catalysis. The principle emerging from these studies is that this conversion is possible in KDO8PS because the metal is not involved in an activation process, but primarily contributes to orienting properly the reactants to lower the activation energy, an action easily mimicked by amino acid side-chains.     

Structural and mechanistic changes along an engineered path from metallo to nonmetallo 3-deoxy-D-manno-octulosonate 8-phosphate synthases

There are two classes of KDO8P synthases characterized respectively by the presence or absence of a metal in the active site. The nonmetallo KDO8PS from Escherichia coli and the metallo KDO8PS from Aquifex aeolicus are the best characterized members of each class. All amino acid residues that make important contacts with the substrates are conserved in both enzymes with the exception of Pro-10, Cys-11, Ser-235, and Gln-237 of the A. aeolicus enzyme, which correspond respectively to Met-25, Asn-26, Pro-252, and Ala-254 in the E. coli enzyme. Interconversion between the two forms of KDO8P synthases can be achieved by substituting the metal-coordinating cysteine of metallo synthases with the corresponding asparagine of nonmetallo synthases, and vice versa. In this report we describe the structural changes elicited by the C11N mutation and by three combinations of mutations (P10M/C11N, C11N/S235P/Q237A, and P10M/C11N/S235P/Q237A) situated along possible evolutionary paths connecting the A. aeolicus and the E. coli enzyme. All four mutants are not capable of binding metal and lack the structural asymmetry among subunits with regard to substrate binding and conformation of the L7 loop, which is typical of A. aeolicus wild-type KDO8PS but is absent in the E. coli enzyme. Despite the lack of the active site metal, the mutant enzymes display levels of activity ranging from 46% to 24% of the wild type. With the sole exception of the quadruple mutant, metal loss does not affect the thermal stability of KDO8PS. The free energy of unfolding in water is also either unchanged or even increased in the mutant enzymes, suggesting that the primary role of the active site metal in A. aeolicus KDO8PS is not to increase the enzyme stability. In all four mutants A5P binding displaces a water molecule located on the si side of PEP. In particular, in the double and triple mutant, A5P binds with the aldehyde carbonyl in hydrogen bond distance of Asn-11, while in the wild type this functional group points away from Cys-11. This alternative conformation of A5P is likely to have functional significance as it resembles the conformation of the acyclic reaction intermediate, which is observed here for the first time in some of the active sites of the triple mutant. The direct visualization of this intermediate by X-ray crystallography confirms earlier mechanistic models of KDO8P synthesis. In particular, the configuration of the C2 chiral center of the intermediate supports a model of the reaction in nonmetallo KDO8PS, in which water attacks an oxocarbenium ion or PEP from the si side of C2. Several explanations are offered to reconcile this observation with the fact that no water molecule is observed at this position in the mutant enzymes in the presence of both PEP and A5P. Significant differences were observed between the wild-type and the mutant enzymes in the Km values for PEP and A5P and in the Kd values for inorganic phosphate and R5P. These differences may reflect an evolutionary adaptation of metallo and nonmetallo KDO8PS's to the cellular concentrations of these metabolites in their respective hosts.     

3-Deoxy-D-manno-octulosonate-8-phosphate synthase catalyzes the C-O bond cleavage of phosphoenolpyruvate

The mechanism of 3-deoxy-D-manno-octulosonate-8-phosphate (KDO8P) synthase was investigated. When [18O]-PEP specifically labeled in the enolic oxygen is a substrate for KDO8P synthase, the 18O is recovered in Pi. This indicates that the KDO8P synthase reaction proceeds with C-O bond cleavage of PEP similar to that observed in the 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase catalyzed condensation of PEP and erythrose-4-phosphate (1). No evidence for a covalent enzyme-PEP intermediate could be obtained. No [32P]-Pi exchange into PEP nor scrambling of bridge 18O to non-bridging positions in [18O]-PEP was observed in the presence or absence of arabinose-5-phosphate or its analog ribose-5-phosphate. Bromopyruvate inactivated KDO8P synthase in a time dependent process. It is likely that bromopyruvate reacts with a functional group at the PEP binding site since PEP, but not arabinose-5-phosphate, protects against inactivation.     

Crystal structures of KDOP synthase in its binary complexes with the substrate phosphoenolpyruvate and with a mechanism-based inhibitor

The crystal structures of 3-deoxy-D-manno-2-octulosonate-8-phosphate synthase (KDOPS) from Escherichia coli complexed with the substrate phosphoenolpyruvate (PEP) and with a mechanism-based inhibitor (K(d) = 0.4 microM) were determined by molecular replacement using X-ray diffraction data to 2.8 and 2.3 A resolution, respectively. Both the KDOPS.PEP and KDOPS.inhibitor complexes crystallize in the cubic space group I23 with cell constants a = b = c = 117.9 and 117.6 A, respectively, and one subunit per asymmetric unit. The two structures are nearly identical, and superposition of their Calpha atoms indicates an rms difference of 0.41 A. The PEP in the KDOPS.PEP complex is anchored to the enzyme in a conformation that blocks its si face and leaves its re face largely devoid of contacts. This results from KDOPS's selective choice of a PEP conformer in which the phosphate group of PEP is extended toward the si face. Furthermore, the structure reveals that the bridging (P-O-C) oxygen atom and the carboxylate group of PEP are not strongly hydrogen-bonded to the enzyme. The resulting high degree of negative charge on the carboxylate group of PEP would then suggest that the condensation step between PEP and D-arabinose-5-phosphate (A5P) should proceed in a stepwise fashion through the intermediacy of a transient oxocarbenium ion at C2 of PEP. The molecular structural results are discussed in light of the chemically similar but mechanistically distinct reaction that is catalyzed by the enzyme 3-deoxy-D-arabino-2-heptulosonate-7-phosphate synthase and in light of the preferred enzyme-bound states of the substrate A5P.     

Probing the role of metal ions in the catalysis of Helicobacter pylori 3-deoxy-D-manno-octulosonate-8-phosphate synthase using a transient kinetic analysis

3-Deoxy-d-manno-2-octulosonate-8-phosphate (KDO8P) synthase catalyzes the net condensation of phosphoenolpyruvate and d-arabinose 5-phosphate to form KDO8P and inorganic phosphate (Pi). Two classes of KDO8P synthases have been identified. The Class I KDO8P synthases (e.g. Escherchia coli KDO8P synthase) catalyze the condensation reaction in a metal-independent fashion, whereas the Class II enzymes (e.g. Aquifex aeolicus) require metal ions for catalysis. Helicobacter pylori (H. pylori) KDO8P synthase, a Zn2+-dependent metalloenzyme, has recently been found to be a Class II enzyme and has a high degree of clinical significance since it is an attractive molecular target for the design of novel antibiotic therapy. Although the presence of a divalent metal ion in Class II KDO8P synthases is essential for catalysis, there is a paucity of mechanistic information on the role of the metal ions and functional differences as compared with Class I enzymes. Using H. pylori KDO8P synthase as a prototypical Class II enzyme, a steady-state and transient kinetic approach was undertaken to understand the role of the metal ion in catalysis and define the kinetic reaction pathway. Metal reconstitution experiments examining the reaction kinetics using Zn2+, Cd2+, Cu2+, Co2+, Mn2+, and Ni2+ yielded surprising results in that the Cd2+ enzyme has the greatest activity. Unlike Class-I KDO8P synthases, the Class II metallo-KDO8P synthases containing Zn2+, Cd2+, Cu2+, and Co2+ show cooperativity. This study presents the first detailed kinetic characterization of a metal-dependent Class II KDO8P synthase and offers mechanistic insight for how the divalent metal ions modulate catalysis through effects on chemistry as well as quaternary protein structure.     

Targeting the role of a key conserved motif for substrate selection and catalysis by 3-deoxy-D-manno-octulosonate 8-phosphate synthase

3-Deoxy-D-manno-octulosonate 8-phosphate synthase (KDO8PS) catalyzes the reaction between three-carbon phosphoenolpyruvate (PEP) and five-carbon d-arabinose 5-phosphate (A5P), generating KDO8P, a key intermediate in the biosynthetic pathway to 3-deoxy-D-manno-octulosonate, a component of the lipopolysaccharide of the Gram-negative bacterial cell wall. Both metal-dependent and metal-independent forms of KDO8PS have been characterized. KDO8PS is evolutionarily and mechanistically related to the first enzyme of the shikimate pathway, the obligately divalent metal ion-dependent 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS) that couples PEP and four-carbon D-erythrose 4-phosphate (E4P) to give DAH7P. In KDO8PS, an absolutely conserved KANRS motif forms part of the A5P binding site, whereas in DAH7PS, an absolutely conserved KPR(S/T) motif accommodates E4P. Here, we have characterized four mutants of this motif (AANRS, KAARS, KARS, and KPRS) in metal-dependent KDO8PS from Acidithiobacillus ferrooxidans and metal-independent KDO8PS from Neisseria meningitidis to test the roles of the universal Lys and the Ala-Asn portion of the KANRS motif. The X-ray structures, determined for the N. meningitidis KDO8PS mutants, indicated no gross structural penalty resulting from mutation, but the subtle changes observed in the active sites of these mutant proteins correlated with their altered catalytic function. (1) The AANRS mutations destroyed catalytic activity. (2) The KAARS mutations lowered substrate selectivity, as well as activity. (3) Replacing KANRS with KARS or KPRS destroyed KDO8PS activity but did not produce a functional DAH7PS. Thus, Lys is critical to catalysis, and other changes are necessary to switch substrate specificity for both the metal-independent and metal-dependent forms of these enzymes.     

Identification of essential histidine residues in 3-deoxy-D-manno-octulosonic acid 8-phosphate synthase: analysis by chemical modification with diethyl pyrocarbonate and site-directed mutagenesis

The enzyme 3-deoxy-D-manno-octulosonic acid 8-phosphate (KDO 8-P) synthase from Escherichia coli that catalyzes the aldol-type condensation of D-arabinose 5-phosphate (A 5-P) and phosphoenolpyruvate (PEP) to give KDO 8-P and inorganic phosphate (P(i)) is inactivated by diethyl pyrocarbonate (DEPC). The inactivation is first-order in enzyme and DEPC. A second-order rate constant of 340 M(-1) min(-1) is obtained at pH 7.6 and 4 degrees C. The rate of inactivation is dependent on pH and the pH-inactivation rate data imply the involvement of an amino acid residue with a pK(a) value of 7.3. KDO 8-P synthase activity is not restored to the DEPC-inactivated enzyme following treatment with hydroxylamine. Complete loss of KDO 8-P synthase activity correlates with the ethoxyformylation of three histidine residues by DEPC. KDO 8-P synthase is protected against DEPC inactivation by PEP and partially protected against inactivation by A 5-P. To provide further evidence for the involvement or role of the histidine residues in the aldol-type condensation catalyzed by KDO 8-P synthase, all six histidines were individually mutated to either glycine or alanine. The kinetic constants for the three mutants H40A, H67G, and H246G were unaffected as compared to the wild type enzyme. In contrast, H241G demonstrates a >10-fold increase in K(M) for both PEP and A 5-P and a 4-fold reduction in k(cat), while H97G demonstrates an increase in K(M) for only A 5-P and a 2-fold reduction in k(cat). The activity of the H202G mutant was too low to be measured accurately but the data obtained indicated an approximate 400-fold reduction in k(cat). Circular dichroism measurements of the wild-type and mutant enzymes indicate modest structural changes in only the fully active H67G and H246G mutants. The H241G mutant is protected against DEPC inactivation by PEP and A 5-P to the same extent as the wild-type enzyme, suggesting that the functionally important H241 may not be located in the vicinity of the substrate binding sites. The H97G mutant is protected by PEP against DEPC inactivation to the same degree as the wild-type enzyme but is no longer protected by A 5-P. In the case of the H202G mutant, both A 5-P and PEP protect the mutant against DEPC inactivation but to different extents from those observed for the wild-type enzyme. The catalytic activity of the H97G mutant is partially restored (20% --> 60% of wild-type activity) in the presence of imidazole, while a minor amount of activity is restored to the H202G mutant (<1% --> 4% of wild-type activity) in the presence of imidazole.     

Mechanistic studies of 3-deoxy-D-manno-2-octulosonate-8-phosphate synthase from Escherichia coli.

The anomeric specificity and the steady-state kinetic mechanism of homogeneous 3-deoxy-D-manno-2-octulosonate-8-phosphate (KDO8P) synthase were investigated. The open-chain 4-deoxy analogue of arabinose-5-phosphate (Ara5P), which is structurally prohibited from undergoing ring closure, was synthesized and tested as a substrate for the synthase. It was found that the analogue functions as a substrate with a similar kcat value to that of the original substrate. The kcat/Km value for the natural substrate is seven-times greater than that of the 4-deoxy analogue. However, taking into account the 9.5% and approximately 1% concentrations of the aldehyde forms of the 4-deoxy analogue and Ara5P in solution, then the 'true' Km values must be in the range 31.5 microM and 0.26 microM, respectively, requiring about a 3 kcal/mol contribution to the binding energy by the 4-hydroxyl group of Ara5P. The data provides evidence that the enzyme acts upon the acyclic form of the natural substrate. The steady-state kinetic study of KDO8P synthase was analyzed via inhibition using the products KDO8P and inorganic phosphate, and D-ribose-5-phosphate as a dead-end inhibitor. First, intersecting lines in double-reciprocal plots of initial-velocity data at substrate concentrations in the micromolar range suggest a sequential mechanism for the enzyme-catalyzed reaction. The inhibition by D-ribose-5-phosphate is competitive for Ara5P and uncompetitive for phosphoenolpyruvate (P-pyruvate). These inhibition patterns are consistent with the model wherein P-pyruvate binding precedes that of Ara5P binding. Furthermore, this order of substrate binding was supported by the observations that KDO8P is a competitive inhibitor for P-pyruvate binding, supporting the concept that KDO8P and P-pyruvate bind to the same enzyme form, and noncompetitively with respect to Ara5P. In addition, the inhibition by inorganic phosphate is noncompetitive with respect to both P-pyruvate and Ara5P, suggesting an apparent ordered release of products such that Pi first, followed by KDO8P. In conclusion, these data suggest a steady-state kinetic mechanism for KDO8P synthase where P-pyruvate binding precedes that of Ara5P, followed by the ordered release of inorganic phosphate and KDO8P.     

A reciprocal single mutation affects the metal requirement of 3-deoxy-D-manno-2-octulosonate-8-phosphate (KDO8P) synthases from Aquifex pyrophilus and Escherichia coli

The enzyme 3-deoxy-d-manno-2-octulosonate-8-phosphate (KDO8P) synthase is metal-dependent in one class of organisms and metal-independent in another. We have used a rapid transient kinetic approach combined with site-directed mutagenesis to characterize the role of the metal ion as well as to explore the catalytic mechanisms of the two classes of enzymes. In the metal-dependent Aquifex pyrophilus KDO8P synthase, Cys11 was replaced by Asn (ApC11N), and in the metal-independent Escherichia coli KDO8P synthase a reciprocal mutation, Asn26 to Cys, was prepared (EcN26C). The ApC11N mutant retained about 10% of the wild-type maximal activity in the absence of metal ions. Addition of divalent metal ions did not affect the catalytic activity of the mutant enzyme and its catalytic efficiency (kcat/Km) was reduced by only approximately 12-fold, implying that the ApC11N KDO8P synthase mutant has become a bone fide metal-independent enzyme. The isolated EcN26C mutant had similar metal content and spectral properties as the metal-dependent wild-type A. pyrophilus KDO8P synthase. EDTA-treated EcN26C retained about 6% of the wild-type activity, and the addition of Mn2+ or Cd2+ stimulated its activity to approximately 30% of the wild-type maximal activity. This suggests that EcN26C KDO8P synthase mutant has properties similar to that of metal-dependent KDO8P synthases. The combined data indicate that the metal ion is not directly involved in the chemistry of the KDO8P synthase catalyzed reaction, but has an important structural role in metal-dependent enzymes in maintaining the correct orientation of the substrates and/or reaction intermediate(s) in the enzyme active site.