Structures of Aquifex aeolicus KDO8P synthase in complex with R5P and PEP, and with a bisubstrate inhibitor: role of active site water in catalysis.

We have determined the crystal structures of the metalloenzyme 3-deoxy-D-manno-octulosonate 8-phosphate (KDO8P) synthase from Aquifex aeolicus in complex with phosphoenolpyruvate (PEP) and ribose 5-phosphate (R5P), and with a bisubstrate inhibitor that mimics the postulated linear reaction intermediate. R5P, which is not a substrate for KDO8P synthase, binds in a manner similar to that of arabinose 5-phosphate (A5P), which is the natural substrate. The lack of reactivity of R5P appears to be primarily a consequence of the loss of a water molecule coordinated to Cd(2+) and located on the si side of PEP. This water molecule is no longer present because it cannot form a hydrogen bond with C2-OH(R5P), which is oriented in a different direction from C2-OH(A5P). The bisubstrate inhibitor binds with its phosphate and phosphonate moieties occupying the positions of the phosphate groups of A5P and PEP, respectively. One of the inhibitor hydroxyls replaces water as a ligand of Cd(2+). The current work supports a mechanism for the synthesis of KDO8P, in which a hydroxide ion on the si side of PEP attacks C2(PEP), forming a tetrahedral-like intermediate with a buildup of negative charge at C3(PEP). The ensuing condensation of C3(PEP) with C1(A5P) would be favored by a proton transfer from the phosphate moiety of PEP to the aldehyde carbonyl of A5P to generate the hydroxyl. Overall, the process can be described as a syn addition of water and A5P to the si side of PEP.

Elucidation of solvent exposure, side-chain reactivity, and steric demands of the trifluoromethionine residue in a recombinant protein.

When incorporated into proteins, fluorinated amino acids have been utilized as 19F NMR probes of protein structure and protein-ligand interactions, and as subtle structural replacements for their parent amino acids which is not possible using the standard 20-amino acid repertoire. Recent investigations have shown the ability of various fluorinated methionines, such as difluoromethionine (DFM) and trifluoromethionine (TFM), to be bioincorporated into recombinant proteins and to be extremely useful as 19F NMR biophysical probes. Interestingly, in the case of the bacteriophage lambda lysozyme (LaL) which contains only three Met residues (at positions 1, 14, and 107), four 19F NMR resonances are observed when TFM is incorporated into LaL. To elucidate the underlying structural reasons for this anomalous observation and to more fully explore the effect of TFM on protein structure, site-directed mutagenesis was used to assign each 19F NMR resonance. Incorporation of TFM into the M14L mutant resulted in the collapse of the two 19F resonances associated with TFM at position 107 into a single resonance, suggesting that when position 14 in wild-type protein contains TFM, a subtle but different environment exists for the methionine at position 107. In addition, 19F and [1H-13C]-HMQC NMR experiments have been utilized with paramagnetic line broadening and K2PtCl4 reactivity experiments to obtain information about the probable spatial position of each Met in the protein. These results are compared with the recently determined crystal structure of LaL and allow for a more detailed structural explanation for the effect of fluorination on protein structure.

Crystal structure of the lytic transglycosylase from bacteriophage lambda in complex with hexa-N-acetylchitohexaose.

The three-dimensional structure of the lytic transglycosylase from bacteriophage lambda, also known as bacteriophage lambda lysozyme, complexed to the hexasaccharide inhibitor, hexa-N-acetylchitohexaose, has been determined by X-ray crystallography at 2.6 A resolution. The unit cell contains two molecules of the lytic transglycosylase with two hexasaccharides bound. Each enzyme molecule is found to interact with four N-acetylglucosamine units from one hexasaccharide (subsites A-D) and two N-acetylglucosamine units from the second hexasaccharide (subsites E and F), resulting in all six subsites of the active site of this enzyme being filled. This crystallographic structure, therefore, represents the first example of a lysozyme in which all subsites are occupied, and detailed protein-oligosaccharide interactions are now available for this bacteriophage lytic transglycosylase. Examination of the active site furthermore reveals that of the two residues that have been implicated in the reaction mechanism of most other c-type lysozymes (Glu35 and Asp52 in hen egg white lysozyme), only a homologous Glu residue is present. The lambda lytic transglycosylase is therefore functionally closely related to the Escherichia coli Slt70 and Slt35 lytic transglycosylases and goose egg white lysozyme which also lack the catalytic aspartic acid.

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.

Histidine 268 in 3-deoxy-D-arabino-heptulosonic acid 7-phosphate synthase plays the same role as histidine 202 in 3-deoxy-D-manno-octulosonic acid 8-phosphate synthase.

The enzyme 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAH 7-P) synthase (Phe) is inactivated by diethyl pyrocarbonate (DEPC). The inactivation is first order with respect to enzyme and DEPC concentrations with a pseudo-second order rate constant of inactivation by DEPC of 4.9 +/- 0.8 m(-1) s(-1) at pH 6.8 and 4 degrees C. The dependence of inactivation on pH and the spectral features of enzyme modified at specific pH values imply that both histidine and cysteine residues are modified, which is confirmed by site-directed mutagenesis. Analysis of the chemical modification data indicates that one histidine is essential for activity. DAH 7-P synthase (Phe) is protected against DEPC inactivation by phosphoenolpyruvate, whereas d-erythrose 4-phosphate offers only minimal protection. The conserved residues H-172, H-207, H-268, and H-304 were individually mutated to glycine. The H304G and H207G mutants retain some level of activity, whereas the H268G and H172G mutants are virtually inactive. A comparison of the circular dichroism spectra of wild-type enzyme and the various mutants demonstrates that H-172 may play a structural role. Comparison of the UV spectra of the H268G and wild-type enzymes saturated with Cu(2+) indicates that the metal-binding site of the H268G mutant resembles that of the wild-type enzyme. The residue H-268 may play a catalytic role based on the site-directed mutagenesis and spectroscopic studies. Cysteine 61 appears to influence the pK(a) of H-268 in the wild-type enzyme. The pK(a) of H-268 increases from 6.0 to 7.0 following mutation of C-61 to glycine.

A metal bridge between two enzyme families. 3-deoxy-D-manno-octulosonate-8-phosphate synthase from Aquifex aeolicus requires a divalent metal for activity.

The enzymes 3-deoxy-d-manno-octulosonic acid-8-phosphate synthase (KDO8PS) and 3-deoxy-d-arabino-heptulosonic acid-7-phosphate synthase (DAHPS) catalyze analogous condensation reactions between phosphoenolpyruvate and d-arabinose 5-phosphate or d-erythrose 4-phosphate, respectively. While several similarities exist between the two enzymatic reactions, classic studies on the Escherichia coli enzymes have established that DAHPS is a metalloenzyme, whereas KDO8PS has no metal requirement. Here, we demonstrate that KDO8PS from Aquifex aeolicus, representing only the second member of the KDO8PS family to be characterized in detail, is a metalloenzyme. The recombinant KDO8PS, as isolated, displays an absorption band at 505 nm and contains approximately 0.4 and 0.2-0.3 eq of zinc and iron, respectively, per enzyme subunit. EDTA inactivates the enzyme in a time- and concentration-dependent manner and eliminates the absorption at 505 nm. The addition of Cu(2+) to KDO8PS produces an intense absorption at 375 nm, while neither Co(2+) nor Ni(2+) produce such an effect. The EDTA-treated enzyme is reactivated by a wide range of divalent metal ions including Ca(2+), Cd(2+), Co(2+), Cu(2+), Fe(2+), Mg(2+), Mn(2+), Ni(2+), and Zn(2+) and is reversibly inhibited by higher concentrations (>1 mm) of certain metals. Analysis of several metal forms of the enzyme by plasma mass spectrometry suggests that the enzyme preferentially binds one, two, or four metal ions per tetramer. These observations strongly suggest that A. aeolicus KDO8PS is a metalloenzyme in vivo and point to a previously unrecognized relationship between the KDO8PS and DAHPS families.

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.

Functional and biochemical characterization of a recombinant 3-Deoxy-D-manno-octulosonic acid 8-phosphate synthase from the hyperthermophilic bacterium Aquifex aeolicus.

The kdsA gene from the hyperthermophilic bacterium Aquifex aeolicus was cloned into a vector for expression in Escherichia coli and the kdsA gene product, 3-deoxy-d-manno-octulosonic acid 8-phosphate synthase (KdsA), was overexpressed under optimized growth conditions. The thermophilic KdsA was purified using an efficient purification procedure including a heat-treatment step. Purified KdsA was shown to catalyze the formation of 3-deoxy-d-manno-octulosonic acid 8-phosphate from phosphoenolpyruvate (PEP) and d-arabinose 5-phosphate (A 5-P) as determined from (1)H NMR analysis of the product. Analytical gel filtration analysis indicated the native enzyme to be oligomeric. KdsA was extremely thermostable, exhibiting maximal activity at 95 degrees C and with half-lives of 1.5 h (90 degrees C), 8.1 h (80 degrees C), and 30.3 h (70 degrees C). KdsA appeared to follow Michaelis-Menton kinetics with K(A5-P)(m) = 8 - 74 microM, K(PEP)(m) = 43-28 microM, and k(cat) = 0.4-2.0 s(-1) between 60 and 90 degrees C.

CNBr/formic acid reactions of methionine- and trifluoromethionine-containing lambda lysozyme: probing chemical and positional reactivity and formylation side reactions by mass spectrometry.

The cyanogen bromide (CNBr)/formic acid cleavage reactions of wild-type and trifluoromethionine (TFM)-containing recombinant lambda lysozyme were studied utilizing ESI and MALDI mass spectrometry. Detailed analysis of the mass spectra of reverse-phase HPLC-purified cleavage fragments produced from treatment of the wild-type and labeled proteins with CNBr indicated cleavage solely of methionyl peptide bonds with no observation of cleavage at TFM. N-Acetyl-TFM was also found to be resistant to reaction with CNBr, in contrast to N-acetyl-methionine. The analysis also indicated differential reactivity among the three methionine positions in the wild-type enzyme. Additionally, formylation of intact enzyme as well as peptide fragments were observed and characterized and indicated that serine, threonine, as well as C-terminal homoserine side chains are partially formylated under standard cleavage protocols.

Investigations of the interactions of saccharides with the lysozyme from bacteriophage lambda.

The bacteriophage lambda R gene has been isolated into an Escherichia coli expression system and the R gene product, a lysozyme, has been overexpressed and purified to homogeneity using an efficient purification procedure. A turbidimetric assay utilizing chloroform-treated E. coli cells has been optimized to assess the bacteriolytic activity of the purified enzyme. Using this assay, oligomers of beta (1 --> 4) N-acetyl-D-glucosamine at high concentrations were shown to inhibit lysozyme but were not cleaved by the enzyme. Differential scanning calorimetry revealed that the thermal denaturation of lysozyme was found to increase in the presence of (GlcNAc)3 and (GlcNAc)5. The lysozyme was also expressed in an E. coli strain auxotrophic for methionine, allowing for the incorporation of [methyl-13C]methionine into the enzyme. An alteration of the [1H-13C]HMQC NMR spectra of the labelled enzyme was observed in the presence of (GlcNAc)5. Commercially available nitrophenyl glycosides did not act as substrates for lambda lysozyme. The results indicate that lambda lysozyme has specific interactions with oligosaccharides of N-acetylglucosamine, but is incapable of hydrolyzing these sugars. The relevance of the structure of peptidoglycan to the activity of lambda lysozyme is discussed.