Refined crystallographic structure of Pseudomonas aeruginosa exotoxin A and its implications for the molecular mechanism of toxicity.

Exotoxin A of Pseudomonas aeruginosa asserts its cellular toxicity through ADP-ribosylation of translation elongation factor 2, predicated on binding to specific cell surface receptors and intracellular trafficking via a complex pathway that ultimately results in translocation of an enzymatic activity into the cytoplasm. In early work, the crystallographic structure of exotoxin A was determined to 3.0 A resolution, revealing a tertiary fold having three distinct structural domains; subsequent work has shown that the domains are individually responsible for the receptor binding (domain I), transmembrane targeting (domain II), and ADP-ribosyl transferase (domain III) activities, respectively. Here, we report the structures of wild-type and W281A mutant toxin proteins at pH 8.0, refined with data to 1.62 A and 1.45 A resolution, respectively. The refined models clarify several ionic interactions within structural domains I and II that may modulate an obligatory conformational change that is induced by low pH. Proteolytic cleavage by furin is also obligatory for toxicity; the W281A mutant protein is substantially more susceptible to cleavage than the wild-type toxin. The tertiary structures of the furin cleavage sites of the wild-type and W281 mutant toxins are similar; however, the mutant toxin has significantly higher B-factors around the cleavage site, suggesting that the greater susceptibility to furin cleavage is due to increased local disorder/flexibility at the site, rather than to differences in static tertiary structure. Comparison of the refined structures of full-length toxin, which lacks ADP-ribosyl transferase activity, to that of the enzymatic domain alone reveals a salt bridge between Arg467 of the catalytic domain and Glu348 of domain II that restrains the substrate binding cleft in a conformation that precludes NAD+ binding. The refined structures of exotoxin A provide precise models for the design and interpretation of further studies of the mechanism of intoxication.

Crystal structure of a lead-dependent ribozyme revealing metal binding sites relevant to catalysis.

The leadzyme is a small RNA motif that catalyzes a site-specific, Pb2+-dependent cleavage reaction. As such, it is an example of a metal-dependent RNA enzyme. Here we describe the X-ray crystallographic structure of the leadzyme, which reveals two independent molecules per asymmetric unit. Both molecules feature an internal loop in which a bulged purine base stack twists away from the helical stem. This kinks the backbone, rendering the phosphodiester bond susceptible to cleavage. The independent molecules have different conformations: one leadzyme copy coordinates Mg2+, whereas the other binds only Ba2+ or Pb2+. In the active site of the latter molecule, a single Ba2+ ion coordinates the 2'-OH nucleophile, and appears to mimic the binding of catalytic lead. These observations allow a bond cleavage reaction to be modeled, which reveals the minimal structural features necessary for catalysis by this small ribozyme.

Crystallographic structures of the hammerhead ribozyme: relationship to ribozyme folding and catalysis.

The hammerhead ribozyme is a small catalytic RNA that cleaves a target phosphodiester bond in a reaction dependent on divalent metal ions. Crystal structures of the hammerhead reveal the tertiary fold of an enzymatic "ground state" of the molecule; however, they do not clarify the catalytic mechanism of the ribozyme, presumably because a significant conformational rearrangement is required to reach an enzymatic transition state. The structural domains seen in the hammerhead can be related to sequence or structural motifs in transfer and ribosomal RNAs, suggesting that they represent tertiary building blocks that will be found in large, complex RNAs.

Structural and mechanistic studies of enolase.

The high-resolution structure of yeast enolase cocrystallized with its equilibrium mixture of substrate and product reveals the stereochemistry of substrate/product binding and therefore the groups responsible for acid/base catalysis and stabilization of the enolate intermediate. Expression and characterization of site-specific mutant forms of the enzyme have confirmed the roles of amino acid side chains in the catalysis of the first and second steps of the reaction. Coordination of both required magnesium ions to the carboxylate of the substrate/product indicates a role for these cations in stabilization of the intermediate.

The enolase superfamily: a general strategy for enzyme-catalyzed abstraction of the alpha-protons of carboxylic acids.

We have discovered a superfamily of enzymes related by their ability to catalyze the abstraction of the alpha-proton of a carboxylic acid to form an enolic intermediate. Although each reaction catalyzed by these enzymes is initiated by this common step, their overall reactions (including racemization, beta-elimination of water, beta-elimination of ammonia, and cycloisomerization) as well as the stereochemical consequences (syn vs anti) of the beta-elimination reactions are diverse. Analysis of sequence and structural similarities among these proteins suggests that all of their chemical reactions are mediated by a common active site architecture modified through evolution to allow the enolic intermediates to partition to different products in their respective active sites via different overall mechanisms. All of these enzymes retain the ability to catalyze the thermodynamically difficult step of proton abstraction. These homologous proteins, designated the "enolase superfamily", include enolase as well as more metabolically specialized enzymes: mandelate racemase, galactonate dehydratase, glucarate dehydratase, muconate-lactonizing enzymes, N-acylamino acid racemase, beta-methylaspartate ammonia-lyase, and o-succinylbenzoate synthase. Comparative analysis of structure-function relationships within the superfamily suggests that carboxyphosphonoenolpyruvate synthase, another member of the superfamily, does not catalyze the reaction proposed in the literature but catalyzes an enolase-like reaction instead. The established and deduced structure-function relationships in the superfamily allow the prediction that other apparent members of the family for which no catalytic functions have yet been assigned will also perform chemistry involving abstraction of the alpha-protons of carboxylic acids.

The structure of nucleotidylated histidine-166 of galactose-1-phosphate uridylyltransferase provides insight into phosphoryl group transfer.

Galactose-1-phosphate uridylyltransferase catalyzes the reaction of UDP-glucose with galactose 1-phosphate to form UDP-galactose and glucose 1-phosphate during normal cellular metabolism. The reaction proceeds through a double displacement mechanism characterized by the formation of a stable nucleotidylated histidine intermediate. This paper describes the preparation of the uridylyl-enzyme complex on the crystalline enzyme from Escherichia coli and its subsequent structure determination by X-ray crystallography. The refined structure has an R-factor of 19.6% (data between 65 and 1.86 A resolution) and reveals modest conformational changes at the active site compared to the inactive UMP/UDP-enzyme complex reported previously [Wedekind, J.E., Frey, P.A., & Rayment, I. (1995) Biochemistry 34, 11049-11061]. In particular, positions of the respective UMP alpha-phosphoryl groups differ by approximately 4 A. Well-defined electron density for the nucleotidylated imidazole supports the existence of a covalent bond between N epsilon 2 of the nucleophile and the alpha-phosphorus of UMP. A hydrogen bond that is conserved in both complexes between His 166 N delta 1 and the carbonyl O of His 164 serves to properly orient the nucleophile and electrostatically stabilize the positively charged imidazolium that results from nucleotidylation. Hydrogen bonds from side-chain Gln 168 to the nonbridging phosphoryl oxygens of the nucleotidyl intermediate appear crucial for the formation and reaction of the uridylyl-enzyme complex as well. The significance of the latter interaction is underscored by the fact that the predominant cause of the metabolic disease galactosemia is the mutation of the corresponding Gln (Gln 188 in humans) to Arg. A comparison to other phosphohistidyl enzymes is described, as well as a revised model for the mechanism of the uridylyltransferase.

A carboxylate oxygen of the substrate bridges the magnesium ions at the active site of enolase: structure of the yeast enzyme complexed with the equilibrium mixture of 2-phosphoglycerate and phosphoenolpyruvate at 1.8 A resolution.

The equilibrium mixture of yeast enolase with substrate, 2-phospho-D-glycerate (2-PGA), and product, phosphoenolpyruvate (P-enolpyruvate), has been crystallized from solutions of poly(ethylene glycol) (PEG) at pH 8.0. Crystals belong to the space group C2 and have unit cell dimensions a = 121.9 A, b = 73.2 A, c = 93.9 A, and beta = 93.3 degrees. The crystals have one dimer per asymmetric unit. Crystals of the equilibrium mixture and of the enolase complex of phosphonoacetohydroxamate (PhAH) are isomorphous, and the structure of the former complex was solved from the coordinates of enolase-(Mg2+)2-PhAH [Wedekind, J. E., Poyner, R. R., Reed, G. H., & Rayment, I. (1994) Biochemistry 33, 9333-9342]. The current crystallographic R-factor is 17.7% for all recorded data (92% complete) to 1.8 A resolution. The electron density map is unambiguous with respect to the positions and liganding of both magnesium ions and with respect to the stereochemistry of substrate/product binding. Both magnesium ions are complexed to functional groups of the substrate/product. The higher affinity Mg2+ coordinates to the carboxylate side chains of Asp 246, Glu 295, and Asp 320, both carboxylate oxygens of the substrate/product, and a water molecule. One of the carboxylate oxygens of the substrate/product also coordinates to the lower affinity Mg2+-thus forming a mu-carboxylato bridge. The other ligands of the second Mg2+ are a phosphoryl oxygen of the substrate/product, two water molecules, and the carbonyl and gamma-oxygens of Ser 39 from the active site loop. The intricate coordination of both magnesium ions to the carboxylate group suggests that both metal ions participate in stabilizing negative charge in the carbanion (aci-carboxylate) intermediate. The epsilon-amino group of Lys 345 is positioned to serve as the base in the forward reaction whereas the carboxylate side chain of Glu 211 is positioned to interact with the 3-OH of 2-PGA. The structure provides a candid view of the catalytic machinery of enolase.

Three-dimensional structure of galactose-1-phosphate uridylyltransferase from Escherichia coli at 1.8 A resolution.

Galactose-1-phosphate uridylyltransferase catalyzes the reversible transfer of the uridine 5'-monophosphoryl moiety of UDP-glucose to the phosphate group of galactose 1-phosphate to form UDP-galactose. This enzyme participates in the Leloir pathway of galactose metabolism, and its absence is the primary cause of the potentially lethal disease galactosemia. The three-dimensional structure of the dimeric enzyme from Escherichia coli complexed with uridine 5'-diphosphate is reported here. The structure was solved by multiple isomorphous replacement and electron density modification techniques and has been refined to 1.8 A resolution. Enzyme subunits consist of a single domain with the topology of a "half-barrel". The barrel staves are formed by nine strands of antiparallel beta-sheet. The barrel axis is approximately parallel to the local dyad that relates each subunit. Two amphipathic helices fill the half-barrel sequestering its hydrophobic interior. An iron atom resides on the outside of the barrel, centered in the subunit interface. Intrasubunit coordination to iron resembles a distorted square pyramid formed by the equatorial ligation of two histidines and a bidentate carboxylate group and a single axial histidine. The subunit interface is stabilized by this coordination and is further characterized by the formation of two intermolecular "mini-sheets" distinct from the strands of the half-barrel. Loops that connect the mini-sheet strands contribute to the formation of the active site, which resides on the external surface of the barrel rim. Loops of the barrel strands are tethered together by a structural zinc atom that orients the local fold in a manner essential for catalysis. In one of the latter loops, S gamma of a cysteine is modified by beta-mercaptoethanol, which prevents the alpha-phosphorus of the nucleotide from access to the nucleophile His166. This conformation does not appear to perturb the interactions to the uracil and ribose moieties as mediated through the side chains of Leu54, Ohe75, Asn77, Asp78, Phe79, and Val108. Several of the latter residues have been implicated in human galactosemia. The present structure explains the deleterious effects of many of those mutations.

Galactose-1-phosphate uridylyltransferase from Escherichia coli, a zinc and iron metalloenzyme.

Galactose-1-P uridylyltransferase purified from Escherichia coli cells grown in enriched medium contains approximately 1.2 mol of tightly bound zinc/mol of subunits as well as variable amounts of iron, up to 0.7 mol/mol of subunits, and no detectable Ca, Cd, Cu, Mo, Ni, Co, Mn, As, Pb, or Se. The chelators, 1,10-phenanthroline, 8-hydroxyquinoline, 8-hydroxyquinoline sulfonate, and 2,2'-bipyridyl remove metal ions from the enzyme and allow the importance of zinc and iron to be evaluated. Dialysis of this enzyme against 2 mM 1,10-phenanthroline, 8-hydroxyquinoline sulfonate, and 2,2'-bipyridyl at millimolar concentrations slowly removes both zinc and iron from the enzyme (t1/2 = 4 days at 24 degrees C) with concomitant loss of enzymatic activity. In chelation experiments utilizing 1,10-phenanthroline, residual enzymatic activity was found to be proportional to the zinc content, to the iron content, and to the sum of zinc and iron. UDP-glucose (0.35 mM) protects the enzyme against loss of metal ions and activity in the presence of 1,10-phenanthroline, whereas glucose-1-P at 70 mM (400 x Km) fails to protect. The enzyme purified from cells grown on a minimal medium containing inorganic salts and glucose supplemented with either ZnSO4 or FeSO4 shows approximately the same level of enzymatic activity as the enzyme from cells grown on enriched medium. These experiments showed that enzymatic activity is supported by either iron or zinc associated with two sites in the enzyme. Enzyme depleted of metal ions by chelators can be partially reactivated by addition of ZnSO4.(ABSTRACT TRUNCATED AT 250 WORDS)

Octahedral coordination at the high-affinity metal site in enolase: crystallographic analysis of the MgII--enzyme complex from yeast at 1.9 A resolution.

The structure of the Mg2+ complex of yeast enolase has been determined from crystals grown in solutions of poly(ethylene glycol) at pH 8.1. Crystals belong to the space group P2(1) and have unit cell dimensions a = 72.5 A, b = 73.2 A, c = 89.1 A, and beta = 104.4 degrees. There is one dimer in the asymmetric unit. The current crystallographic R-factor is 19.0% for all recorded data to 1.9 A resolution. The electron density indicates a hexacoordinate Mg2+ at the high-affinity cation binding site. The octahedral coordination sphere consists of a meridional arrangement of three carboxylate oxygens from the side chains of Asp 246, Asp 320, and Glu 295, and three well-ordered water molecules. Octahedral coordination is the preferred geometry for alkaline earth metal ions in complexes with oxygen donor groups. In previous crystallographic studies of enolase, Zn2+ and Mg2+ complexes at the high-affinity site were reported to exist in trigonal bipyramidal coordination. This geometry was suggested to enhance the electrophilicity of the metal ion and promote rapid ligand exchange [Lebioda, L., & Stec, B. (1989) J. Am. Chem. Soc. 111, 8511-8513]. The octahedral arrangement of carboxylate and water ligands in the MgII-enolase complex determined here is most consistent with reports of the Mn2+ and Mg2+ coordination complexes of mandelate racemase and muconate lactonizing enzyme. These latter enzymes have alpha/beta-barrel folds comparable to enolase.(ABSTRACT TRUNCATED AT 250 WORDS)

Chelation of serine 39 to Mg2+ latches a gate at the active site of enolase: structure of the bis(Mg2+) complex of yeast enolase and the intermediate analog phosphonoacetohydroxamate at 2.1-A resolution.

The structure of a new crystal form of enolase from bakers' yeast has been solved to 2.1-A resolution. Crystals were grown from poly(ethylene glycol) and KCl at pH 8.2 in the presence of Mg2+ and a reaction intermediate analog, phosphonoacetohydroxamate (PhAH). Crystals belong to space group C2; have unit cell dimensions a = 123.5 A, b = 73.9 A, and c = 94.8 A with beta = 93.3 degrees; and contain one dimer per asymmetric unit. The structure was solved by molecular replacement from the X-ray coordinates of apoenolase [Stec, B., & Lebioda, L. (1990) J. Mol. Biol. 211, 235-248]. Both essential divalent metal ions are observed to be complexed with the inhibitor. The two Mg2+ ions are 4.05 A apart and are bridged by a mu-oxyl ligand from the carbonyl moiety of PhAH. The "high-affinity" Mg2+ coordinates to the carboxylate side chains of Asp 246, Glu 295, and Asp 320, one water molecule, and the hydroxamate and carbonyl oxygens of PhAH. The second Mg2+ coordinates to a phosphonyl oxygen, two water molecules, and the mu-bridge carbonyl oxygen of PhAH. Coordination schemes with respect to PhAH and water ligands are fully consistent with those of the Mn2+ complexes determined spectroscopically [Poyner, R.R., & Reed, G. H. (1992) Biochemistry 31, 7166-7173]. Remaining ligands for the second Mg2+ are the carbonyl oxygen and gamma-oxygen of Ser 39. Chelation of this Ser residue to Mg2+ effectively "latches" a flexible loop extending from Gly 37 through His 43 and closes off the entrance to the active site. The position of the second Mg2+ in the active site provides new insight into the stereochemistry of substrate binding.

Crystallization and preliminary crystallographic analysis of galactose-1-phosphate uridylyltransferase from Escherichia coli.

Galactose-1-phosphate uridylyltransferase catalyzes the formation of UDP-galactose during normal cellular metabolism, making it an essential enzyme in all cells. The enzyme from Escherichia coli has been crystallized at pH 5.9 in the presence of phenyl-UDP (P(1)-5'-uridyl-P(2)-phenyl diphosphate), a substrate analog, using PEG 10 000 in combination with Li(2)SO(4) and NaCl. Crystals belong to space group P2(1)2(1)2 with unit-cell dimensions a = 58.6, b = 217.6 and c = 69.6 A. There is one dimer or two subunits in the asymmetric unit. Crystals are relatively insensitive to X-ray radiation and diffract beyond 2.5 A resolution. A low-resolution native data set has been recorded.