Lysine 22 in UDP-N-acetylglucosamine enolpyruvyl transferase from Enterobacter cloacae is crucial for enzymatic activity and the formation of covalent adducts with the substrate phosphoenolpyruvate and the antibiotic fosfomycin.

UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) catalyzes the first committed step in the biosynthesis of the bacterial cell wall component peptidoglycan. The enzyme is the target of the antibiotic fosfomycin. A lysine residue (K22), strictly conserved in MurAs and the structurally and mechanistically related 5-enolpyruvylshikimate 3-phosphate synthases (EPSPS), is located near the active center of the enzyme. This residue is thought to be involved directly in the binding of the substrate phosphoenolpyruvate (PEP) and also to participate in the conformational change leading to the formation of the catalytically competent enzyme complex. Using site-directed mutagenesis, we have replaced this lysine with arginine (K22R), valine (K22V), and glutamate (K22E). These mutant proteins were expressed, purified, and characterized in comparison to wild-type MurA and a previously described inactive C115S mutant protein. It was found that all three K22 mutant proteins had less than 0.5% of the wild-type activity. Using isothermal titration calorimetry, it could be shown that the binding parameters for the UDP-sugar nucleotide substrate are not affected by the mutations, except for the K22E mutant protein. Similarly, binding of PEP was found to be unaffected in the K22 mutant proteins as demonstrated by tryptophan fluorescence quench titrations. On the other hand, the level of formation of a covalent adduct with either PEP or fosfomycin with the thiol group of cysteine 115 was diminished. The propensity to form an adduct with PEP decreased in the following order: wild type > K22R > K22V > K22E. A comparable effect was found on the formation of the inhibitory covalent adduct of MurA and the antibiotic fosfomycin. These results are discussed in terms of an involvement of lysine 22 in a conformational change of MurA.     

Role of the loop containing residue 115 in the induced-fit mechanism of the bacterial cell wall biosynthetic enzyme MurA

The induced-fit mechanism in Enterobacter cloacae MurA has been investigated by kinetic studies and X-ray crystallography. The antibiotic fosfomycin, an irreversible inhibitor of MurA, induced a structural change in UDP-N-acetylglucosamine (UDPGlcNAc)-liganded enzyme with a time dependence similar to that observed for the inactivation progress. The mechanism of action of fosfomycin on MurA appeared to be of the bimolecular type, the overall rate constants of inactivation and structural change being = 104 M(-1) s(-1) and = 85 M(-1) s(-1), respectively. Fosfomycin as well as the second MurA substrate, phosphoenolpyruvate (PEP), are known to interact with the side chain of Cys115. Like wild-type MurA, the catalytically inactive single-site mutant protein Cys115Ser structurally interacted with UDPGlcNAc in a rapidly reversible reaction. However, in contrast to wild-type enzyme, binding of PEP to mutant protein induced a rate-limited, biphasic structural change. Fosfomycin did not affect the structure of the mutant protein. The crystal structure of unliganded Cys115Ser MurA at 1.9 A resolution revealed that the overall conformation of the loop comprising residues 112-121 is not influenced by the mutation. However, other than Cys115 in wild-type MurA, Ser115 exhibits two distinct side-chain conformations. A detailed view on the loop revealed the existence of an elaborate hydrogen-bonding network mainly supplied by water molecules, presumably stabilizing its conformation in the unliganded state. The comparison between the known crystal structures of MurA, together with the kinetic data obtained, suggest intermediate conformational states in the MurA reaction, in which the loop undergoes multiple structural changes upon ligand binding.     

Determination of the pKa value of C115 in MurA (UDP-N-acetylglucosamine enolpyruvyltransferase) from Enterobacter cloacae

The enzyme UDP-N-acetylglucosamine (UDP-NAG) enolpyruvyltransferase (MurA) catalyzes the formation of enolpyruvyl-UDP-NAG, a precursor in peptidoglycan biosynthesis. The residue at position 115 in MurA has been proposed to act as a general acid in the enzymatic reaction. This is also the primary site of action of the antibiotic fosfomycin. In this paper, the pK(a) of Cys-115 has been determined to be 8.3, by titration of Enterobacter cloacae MurA with the alkylating agent iodoacetamide as a function of pH. Use of site-directed mutagenesis has established that only C115 is essential for catalysis, and the three other cysteine residues (C251, C354, and C381) are nonessential. Mass spectrometric analysis demonstrated that C115 is not alkylated at pH <7, but is alkylated significantly at pH >7. Measurement of the enzymatic inhibition by iodoacetamide as a function of pH showed maximum inhibition at pH >9, with a second-order rate constant of inhibition of 44 M(-)(1) s(-)(1) at pH 10. The presence of either one of the substrates did not influence the inactivation behavior, while the presence of both substrates resulted in a 5-fold reduction in the extent of alkylation. The covalent species that results from PEP bound to C115 of MurA exhibited 50-100-fold increased resistance against alkylation by iodoacetamide. These results imply that C115 is appreciably protonated at physiological pH and, therefore, is capable of acting as a proton donor in the enzyme-catalyzed reaction. However, it also implies that C115 is appreciably deprotonated at physiological pH also, whereupon the resultant thiolate nucleophile may play an important role in the formation of the covalent O-phosphothioketal species, whose role in catalysis is yet to be established.     

Asparagine 23 and aspartate 305 are essential residues in the active site of UDP-N-acetylglucosamine enolpyruvyl transferase from Enterobacter cloacae

UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) catalyzes the transfer of the intact enolpyruvyl moiety of phosphoenolpyruvate (PEP) to the 3'-hydroxyl group of UDP-N-acetylglucosamine (UDPNAG). This reaction constitutes the first committed step in the biosynthesis of the bacterial cell wall component peptidoglycan (murein). The transfer reaction involves the nucleophilic attack of the 3'-hydroxyl group of UDPNAG at the C-2 of PEP. The three-dimensional structure of MurA complexed with UDPNAG revealed an aspartate residue (D305 in the En. cloacae sequence) close to the 3'-hydroxyl group of UDPNAG, suggesting that it may act as an acid-base catalyst in the active center of the enzyme. In addition to aspartate 305, asparagine 23 also interacts with the 3'-hydroxyl group; however, its role in catalysis or binding of the UDPNAG substrate is unclear. To gain information on the role of these two amino acids in the MurA-catalyzed reaction we have exchanged D305 for alanine, cysteine, histidine, and glutamate, and N23 for alanine and serine using site-directed mutagenesis. While the D305 alanine, cysteine, and histidine mutant proteins do not have detectable enzymatic activity, the D305E mutant protein exhibits a low residual activity (ca. 0.1% of the wild-type enzyme). Unlike with wild-type MurA, no exothermic signal was obtained when the D305A and -E mutant proteins were titrated with UDPNAG, demonstrating that the affinity of the sugar nucleotide substrate is reduced as a result of the amino acid exchange. The reduced affinity to UDPNAG leads to a lower propensity of C115 to form either the O-phosphothioketal with PEP or the thioether with the antibiotic fosfomycin. These findings emphasize the dual role of D305 as a general base and an essential binding partner to UDPNAG in the active site of MurA. Similarly, the two N23 mutant proteins showed a much lower catalytic activity although binding of UDPNAG was not as much affected as in the case of the D305 mutant proteins. This result indicates that this amino acid residue is mainly involved in stabilization of transition states.     

A novel inhibitor that suspends the induced fit mechanism of UDP-N-acetylglucosamine enolpyruvyl transferase (MurA)

MurA (UDP-N-acetylglucosamine enolpyruvyl transferase, EC 2.5.1.7) catalyzes the first committed step in the synthesis of the bacterial cell wall. It is the target of the naturally occurring, broad-spectrum antibiotic fosfomycin. Fosfomycin, an epoxide, is a relatively poor drug because an ever-increasing number of bacteria have developed resistance to fosfomycin. Thus, there is a critical need for the development of novel drugs that target MurA by a different molecular mode of action. We have identified a new scaffold of potent MurA inhibitors, derivatives of 5-sulfonoxy-anthranilic acid, using high-throughput screening. T6361 and T6362 are competitive inhibitors of MurA with respect to the first substrate, UDP-N-acetylglucosamine (UNAG), with a K(i) of 16 microM. The crystal structure of the MurA.T6361 complex at 2.6 angstrom resolution, together with fluorescence data, revealed that the inhibitor targets a loop, Pro112 to Pro121, that is crucial for the structural changes of the enzyme during catalysis. Thus, this new class of MurA inhibitors is not active site-directed but instead obstructs the transition from the open (unliganded) to the closed (UNAG-liganded) enzyme form. The results provide evidence for the existence of a MurA.UNAG collision complex that may be specifically targeted by small molecules different from ground-state analogs of the enzymatic reaction.     

Evidence that the fosfomycin target Cys115 in UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) is essential for product release

MurA (UDP-N-acetylglucosamine enolpyruvyl transferase, EC 2.5.1.7) is an essential enzyme in the biosynthesis of the peptidoglycan layer of the bacterial cell. It provides an attractive template for the design of novel antibiotic drugs and is the target of the naturally occurring antibiotic fosfomycin, which covalently attaches to Cys115 in the active site of the enzyme. Mutations of Cys115 to Asp exist in pathogens such as Mycobacteria or Chlamydia rendering these organisms resistant to fosfomycin. Thus, there is a need for the elucidation of the role of this cysteine in the MurA reaction. We determined the x-ray structure of the C115S mutant of Enterobacter cloacae MurA, which was crystallized in the presence of the substrates of MurA. The structure depicts the product state of the enzyme with enolpyruvyl-UDP-N-acetylglucosamine and inorganic phosphate trapped in the active site. Kinetic analysis revealed that the Cys-to-Ser mutation results in an enzyme that appears to perform a single turnover of the reaction. Opposing the common view of Cys115 as a key residue in the chemical reaction of enolpyruvyl transfer, we now conclude that the wild-type cysteine is essential for product release only. On the basis of a detailed comparison of the product state with the intermediate state and an unliganded state of MurA, we propose that dissociation of the products is an ordered event with inorganic phosphate leaving first. Phosphate departure appears to trigger a suite of conformational changes, which finally leads to opening of the two-domain structure of MurA and the release of the second product enolpyruvyl-UDP-N-acetylglucosamine.     

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.     

UDP-N-acetylmuramic acid (UDP-MurNAc) is a potent inhibitor of MurA (enolpyruvyl-UDP-GlcNAc synthase)

Purified recombinant MurA (enolpyruvyl-UDP-GlcNAc synthase) overexpressed in Escherichia coli had significant amounts of UDP-MurNAc (UDP-N-acetylmuramic acid) bound after purification. UDP-MurNAc is the product of MurB, the next enzyme in peptidoglycan biosynthesis. About 25% of MurA was complexed with UDP-MurNAc after five steps during purification that should have removed it. UDP-MurNAc isolated from MurA was identified by mass spectrometry, NMR analysis, and comparison with authentic UDP-MurNAc. Subsequent investigation showed that UDP-MurNAc bound to MurA tightly, with K(d,UDP)(-)(MurNAc) = 0.94 +/- 0.04 microM, as determined by fluorescence titrations using ANS (8-anilino-1-naphthalenesulfonate) as an exogenous fluorophore. UDP-MurNAc binding was competitive with ANS and phosphate, the second product of MurA, and it inhibited MurA. The inhibition patterns were somewhat ambiguous, likely being competitive with the substrate PEP (phosphoenolpyruvate) and either competitive or noncompetitive with respect to the substrate UDP-GlcNAc (UDP-N-acetylglucosamine). These results indicate a possible role for UDP-MurNAc in regulating the biosynthesis of nucleotide precursors of peptidoglycan through feedback inhibition. Previous studies indicated that UDP-MurNAc binding to MurA was not tight enough to be physiologically relevant; however, this was likely an artifact of the assay conditions.     

Thermodynamic characterization of ligand-induced conformational changes in UDP-N-acetylglucosamine enolpyruvyl transferase

The binding of UDP-N-acetylglucosamine (UDPNAG) to the enzyme UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) was studied in the absence and presence of the antibiotic fosfomycin by isothermal titration calorimetry. Fosfomycin binds covalently to MurA in the presence of UDPNAG and also in its absence as demonstrated by MALDI mass spectrometry. The covalent attachment of fosfomycin affects the thermodynamic parameters of UDPNAG binding significantly: In the absence of fosfomycin the binding of UDPNAG is enthalpically driven (DeltaH = -35.5 kJ mol(-1) at 15 degrees C) and opposed by an unfavorable entropy change (DeltaS = -25 J mol(-1) K(-1)). In the presence of covalently attached fosfomycin the binding of UDPNAG is entropically driven (DeltaS = 187 J mol(-1)K(-1) at 15 degrees C) and associated with unfavorable changes in enthalpy (DeltaH = 28.8 kJ mol(-1)). Heat capacities for UDPNAG binding in the absence or presence of fosfomycin were -1.87 and -2.74 kJ mol(-1) K(-1), respectively, indicating that most ( approximately 70%) of the conformational changes take place upon formation of the UDPNAG-MurA binary complex. The major contribution to the heat capacity of ligand binding is thought to be due to changes in the solvent-accessible surface area. However, associated conformational changes, if any, also contribute to the experimentally measured magnitude of the heat capacity. The changes in solvent-accessible surface area were calculated from available 3D structures, yielding a DeltaC(p) of -1.3 kJ mol(-1) K(-1); i.e., the experimentally determined heat capacity exceeds the calculated one. This implies that other thermodynamic factors exert a large influence on the heat capacity of protein-ligand interactions.     

Structural basis of biological nitrile reduction

The enzyme QueF catalyzes the reduction of the nitrile group of 7-cyano-7-deazaguanine (preQ(0)) to 7-aminomethyl-7-deazaguanine (preQ(1)), the only nitrile reduction reaction known in biology. We describe here two crystal structures of Bacillus subtilis QueF, one of the wild-type enzyme in complex with the substrate preQ(0), trapped as a covalent thioimide, a putative intermediate in the reaction, and the second of the C55A mutant in complex with the substrate preQ(0) bound noncovalently. The QueF enzyme forms an asymmetric tunnel-fold homodecamer of two head-to-head facing pentameric subunits, harboring 10 active sites at the intersubunit interfaces. In both structures, a preQ(0) molecule is bound at eight sites, and in the wild-type enzyme, it forms a thioimide covalent linkage to the catalytic residue Cys-55. Both structural and transient kinetic data show that preQ(0) binding, not thioimide formation, induces a large conformational change in and closure of the active site. Based on these data, we propose a mechanism for the activation of the Cys-55 nucleophile and subsequent hydride transfer.     

Insights into the structure, solvation, and mechanism of ArsC arsenate reductase, a novel arsenic detoxification enzyme.

BACKGROUND: In Escherichia coli bearing the plasmid R773, resistance to arsenite, arsenate, antimonite, and tellurite is conferred by the arsRDABC plasmid operon that codes for an ATP-dependent anion pump. The product of the arsC gene, arsenate reductase (ArsC), is required to efficiently catalyze the reduction of arsenate to arsenite prior to extrusion. RESULTS: Here, we report the first X-ray crystal structures of ArsC at 1.65 A and of ArsC complexed with arsenate and arsenite at 1.26 A resolution. The overall fold is unique. The native structure shows sulfate and sulfite ions binding in the active site as analogs of arsenate and arsenite. The covalent adduct of arsenate with Cys-12 in the active site of ArsC, which was analyzed in a difference map, shows tetrahedral geometry with a sulfur-arsenic distance of 2.18 A. However, the corresponding adduct with arsenite binds as a hitherto unseen thiarsahydroxy adduct. Finally, the number of bound waters (385) in this highly ordered crystal structure approaches twice the number expected at this resolution for a structure of 138 ordered residues. CONCLUSIONS: Structural information from the adduct of ArsC with its substrate (arsenate) and with its product (arsenite) together with functional information from mutational and biochemical studies on ArsC suggest a plausible mechanism for the reaction. The exceptionally well-defined water structure indicates that this crystal system has precise long-range order within the crystal and that the upper limit for the number of bound waters in crystal structures is underestimated by the structures in the Protein Data Bank.     

Characterization of covalent Ene adduct intermediates in "hydride equivalent" transfers in a dihydropyridine model for NADH reduction reactions

A study of the reactions of an NADH model, 1,4-di(trimethylsilyl)-1,4-dihydropyridine, 7, with a series of α,β-unsaturated cyano and carbonyl compounds has produced the first direct evidence for an obligatory covalent adduct between a dihydropyridine and substrate in a reduction reaction. The reactions were monitored by NMR spectroscopy. In all reactions studied, the covalent adduct was the first new species detected and its decomposition to form products could be observed. Concentrations of adducts were sufficiently high at steady-state that their structures could be determined directly from NMR spectra of the reaction mixtures; adduct structures are those expected from an Ene reaction between 7 and the substrate. This first reaction step results in transfer of the C(4) hydrogen nucleus of 7 to the substrate and formation of a covalent bond between C(2) of the dihydropyridine ring and the substrate α-atom. Discovery of these Ene-adduct intermediates completes the spectrum of mechanisms observed in NADH model reactions to span those with free radical intermediates, no detectable intermediates and now covalent intermediates. The geometry of the transition state for formation of the Ene adduct is compared with those of theoretical transition state models and crystal structures of enzyme-substrate/inhibitor complexes to suggest a relative orientation for the dihydropyridine ring and the substrate in an initial cyclic transition state that is flexible enough to accommodate all observed mechanistic outcomes.     

Role of K22 and R120 in the covalent binding of the antibiotic fosfomycin and the substrate-induced conformational change in UDP-N-acetylglucosamine enolpyruvyl transferase.

UDP-N-acetylglucosamine enolpyruvyl transferase (MurA), catalyzes the first step in the biosynthesis of peptidoglycan, involving the transfer of the intact enolpyruvyl moiety from phosphoenolpyruvate to the 3'-hydroxyl group of UDP-N-acetylglucosamine (UDPNAG). The enzyme is irreversibly inhibited by the antibiotic fosfomycin. The inactivation is caused by alkylation of a highly conserved cysteine residue (C115) that participates in the binding of phosphoenolpyruvate. The three-dimensional structure of the enzyme suggests that two residues may play a decisive role in fosfomycin binding: K22 and R120. To investigate the role of these residues, we have generated the K22V, K22E, K22R and R120K single mutant proteins as well as the K22V/R120K and K22V/R120V double mutant proteins. We demonstrated that the K22R mutant protein behaves similarly to wild-type enzyme, whereas the K22E mutant protein failed to form the covalent adduct. On the other hand, the K22V mutant protein requires the presence of UDPNAG for the formation of the adduct indicating that UDPNAG plays a crucial role in the organization of productive interactions in the active site. This model receives strong support from heat capacity changes observed for the K22V/R120K and R120K mutant proteins: in both mutant proteins, the heat capacity changes are markedly reduced indicating that their ability to form a closed protein conformation is impeded due to the R120K exchange.     

Mechanism of the EPSP synthase catalyzed reaction: evidence for the lack of a covalent carboxyvinyl intermediate in catalysis

In order to detect covalent reaction intermediates in the 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase reaction, we have investigated the interaction of EPSP synthase with the reaction product EPSP. An exchange of EPSP-methylene protons could be demonstrated by incubating EPSPS with EPSP in D2O. Since trace amounts of contaminating Pi would lead to reversal of EPSPS reaction and hence methylene proton exchange, we added pyruvate kinase, ADP, Mg++ and K+. Under these conditions, any contaminating Pi that is converted to PEP is trapped as ATP. No exchange of EPSP protons with those of the solvent could be detected in the presence of this trap system, suggesting that enzyme-bound EPSP is unable to form a covalent tetrahedral complex. Incorporation of [14C] from [14C]-S3P and [14C]-PEP into EPSP could be detected, but only in the absence of a PEP (or Pi) trap system. This indicates that for the exchange reaction, Pi is required, and also indicates the absence of a covalent intermediate, unless the carboxyvinyl-enzyme-bound S3P is completely restricted from exchange.     

Characterization and inhibition study of MurA enzyme by capillary electrophoresis

A capillary electrophoresis-based enzyme assay for UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) is described. This method, based on UV detection, provides baseline separation of one of the reaction products, enolpyruvyluridine 5'-diphospho-N-acetylglucosamine (EP-UDP-GlcNAc), from substrates phosphoenolpyruvate (PEP) and uridine 5'-diphospho-N-acetylglucosamine (UDP-GlcNAc) within 4 min. The other product, phosphate, is not detectable by UV at 200 nm. Quantitation of individual components, substrates or product, can be accomplished based on the separated peaks. This methodology was used to determine the Michaelis constant, Km, and product formation rate constant, Kcat, for MurA. Additionally, the CE method was used to evaluate the inhibition effects on MurA using one specific compound as an example. By following similar procedures, the apparent Km values in the presence of different inhibitor concentrations were determined. The inhibition constant, Ki, can be determined from these apparent Km values. In addition, this CE method can be used to study the inhibition mechanism. The principle of this approach is generally applicable to other enzyme studies.     

A new view of the mechanisms of UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) and 5-enolpyruvylshikimate-3-phosphate synthase (AroA) derived from X-ray structures of their tetrahedral reaction intermediate states

UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) and 5-enolpyruvylshikimate-3-phosphate synthase (AroA) constitute the small enzyme family of enolpyruvyl transferases, which catalyze the chemically unusual reaction of enolpyruvyl transfer. MurA catalyzes the first step in the biosynthesis of the bacterial cell wall; AroA is the sixth enzyme of the shikimate pathway leading to the synthesis of aromatic compounds in numerous microorganisms and plants. Because both metabolic pathways are absent from mammals but essential for the growth of microorganisms, MurA and AroA are attractive targets for the development of novel antimicrobial drugs. We have determined the x-ray structures of the D305A mutant of Enterobacter cloacae MurA and the D313A mutant of Escherichia coli AroA, both of which crystallized in the presence of their substrates. The structures depict the tetrahedral reaction intermediate states of the enzymes and prove that, without the aspartate side chain, the overall addition-elimination reaction in both enzymes is halted after the addition step. The presented structures lead to a new view of the catalytic mechanism and, moreover, provide an ideal starting point for the rational design of potent inhibitors of MurA and AroA.     

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.     

The high-resolution structure of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase reveals a twist in the plane of bound phosphoenolpyruvate

3-Deoxy-D-arabino-heptulosonate-7-phosphate synthase (DAHPS), the first enzyme of the aromatic biosynthetic pathway in microorganisms and plants, catalyzes the aldol-like condensation of phosphoenolpyruvate (PEP) and D-erythrose 4-phosphate (E4P) with the formation of DAHP. The native and the selenomethionine-substituted forms of the phenylalanine-regulated isozyme [DAHPS(Phe)] from Escherichia coli were crystallized in complex with PEP and a metal cofactor, Mn(2+), but the crystals displayed disorder in their unit cells, preventing satisfactory refinement. However, the crystal structure of the E24Q mutant form of DAHPS(Phe) in complex with PEP and Mn(2+) has been determined at 1.75 A resolution. Unlike the tetrameric wild-type enzyme, the E24Q enzyme is dimeric in solution, as a result of the mutational perturbation of four intersubunit salt bridges that are critical for tetramer formation. The protein chain conformation and subunit arrangement in the crystals of E24Q and wild-type DAHPS are very similar. However, the interaction of Mn(2+) and PEP in the enzymatically active E24Q mutant complex differs from the Pb(2+)-PEP and Mn(2+)-phosphoglycolate interactions in two enzymatically inactive wild-type complexes whose structures have been determined previously. The geometry of PEP bound in the active site of the E24Q enzyme deviates from planarity due to a 30 degrees twist of the carboxylate plane relative to the enol plane. In addition, seven water molecules are within contact distance of PEP, two of which are close enough to its C2 atom to serve as the nucleophile required in the reaction.     

Dopamine-derived quinones affect the structure of the redox sensor DJ-1 through modifications at Cys-106 and Cys-53

The physiological role of DJ-1, a protein involved in familial Parkinson disease is still controversial. One of the hypotheses proposed indicates a sensor role for oxidative stress, through oxidation of a conserved cysteine residue (Cys-106). The association of DJ-1 mutations with Parkinson disease suggests a loss of function, specific to dopaminergic neurons. Under oxidative conditions, highly reactive dopamine quinones (DAQs) can be produced, which can modify cysteine residues. In cellular models, DJ-1 was found covalently modified by dopamine. We analyzed the structural modifications induced on human DJ-1 by DAQs in vitro. We described the structural perturbations induced by DAQ adduct formation on each of the three cysteine residues of DJ-1 using specific mutants. Cys-53 is the most reactive residue and forms a covalent dimer also in SH-SY5Y DJ-1-transfected cells, but modification of Cys-106 induces the most severe structural perturbations; Cys-46 is not reactive. The relevance of these covalent modifications to the several functions ascribed to DJ-1 is discussed in the context of the cell response to a dopamine-derived oxidative insult.     

Crystal structures of active fully assembled substrate- and product-bound complexes of UDP-N-acetylmuramic acid:L-alanine ligase (MurC) from Haemophilus influenzae

UDP-N-acetylmuramic acid:L-alanine ligase (MurC) catalyzes the addition of the first amino acid to the cytoplasmic precursor of the bacterial cell wall peptidoglycan. The crystal structures of Haemophilus influenzae MurC in complex with its substrate UDP-N-acetylmuramic acid (UNAM) and Mg(2+) and of a fully assembled MurC complex with its product UDP-N-acetylmuramoyl-L-alanine (UMA), the nonhydrolyzable ATP analogue AMPPNP, and Mn(2+) have been determined to 1.85- and 1.7-A resolution, respectively. These structures reveal a conserved, three-domain architecture with the binding sites for UNAM and ATP formed at the domain interfaces: the N-terminal domain binds the UDP portion of UNAM, and the central and C-terminal domains form the ATP-binding site, while the C-terminal domain also positions the alanine. An active enzyme structure is thus assembled at the common domain interfaces when all three substrates are bound. The MurC active site clearly shows that the gamma-phosphate of AMPPNP is positioned between two bound metal ions, one of which also binds the reactive UNAM carboxylate, and that the alanine is oriented by interactions with the positively charged side chains of two MurC arginine residues and the negatively charged alanine carboxyl group. These results indicate that significant diversity exists in binding of the UDP moiety of the substrate by MurC and the subsequent ligases in the bacterial cell wall biosynthesis pathway and that alterations in the domain packing and tertiary structure allow the Mur ligases to bind sequentially larger UNAM peptide substrates.