Domenico L. Gatti

Structure of the ArsA ATPase: the catalytic subunit of a heavy metal resistance pump

Active extrusion is a common mechanism underlying detoxification of heavy metals, drugs and antibiotics in bacteria, protozoa and mammals. In Escherichia coli, the ArsAB pump provides resistance to arsenite and antimonite. This pump consists of a soluble ATPase (ArsA) and a membrane channel (ArsB). ArsA contains two nucleotide‐binding sites (NBSs) and a binding site for arsenic or antimony. Binding of metalloids stimulates ATPase activity. The crystal structure of ArsA reveals that both NBSs and the metal‐binding site are located at the interface between two homologous domains. A short stretch of residues connecting the metal‐binding site to the NBSs provides a signal transduction pathway that conveys information on metal occupancy to the ATP hydrolysis sites. Based on these structural features, we propose that the metal‐binding site is involved directly in the process of vectorial translocation of arsenite or antimonite across the membrane. The relative positions of the NBS and the inferred mechanism of allosteric activation of ArsA provide a useful model for the interaction of the catalytic domains in other transport ATPases.

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.

Protein Phosphorylation and Prevention of Cytochrome Oxidase Inhibition by ATP: Coupled Mechanisms of Energy Metabolism Regulation

Rapid regulation of oxidative phosphorylation is crucial for mitochondrial adaptation to swift changes in fuels availability and energy demands. An intramitochondrial signaling pathway regulates cytochrome oxidase (COX), the terminal enzyme of the respiratory chain, through reversible phosphorylation. We find that PKA-mediated phosphorylation of a COX subunit dictates mammalian mitochondrial energy fluxes and identify the specific residue (S58) of COX subunit IV-1 (COXIV-1) that is involved in this mechanism of metabolic regulation. Using protein mutagenesis, molecular dynamics simulations, and induced fit docking, we show that mitochondrial energy metabolism regulation by phosphorylation of COXIV-1 is coupled with prevention of COX allosteric inhibition by ATP. This regulatory mechanism is essential for efficient oxidative metabolism and cell survival. We propose that S58 COXIV-1 phosphorylation has evolved as a metabolic switch that allows mammalian mitochondria to rapidly toggle between energy utilization and energy storage.

Protein and ligand dynamics in 4-hydroxybenzoate hydroxylase.

para-Hydroxybenzoate hydroxylase catalyzes a two-step reaction that demands precise control of solvent access to the catalytic site. The first step of the reaction, reduction of flavin by NADPH, requires access to solvent. The second step, oxygenation of reduced flavin to a flavin C4a-hydroperoxide that transfers the hydroxyl group to the substrate, requires that solvent be excluded to prevent breakdown of the hydroperoxide to oxidized flavin and hydrogen peroxide. These conflicting requirements are met by the coordination of multiple movements involving the protein, the two cofactors, and the substrate. Here, using the R220Q mutant form of para-hydroxybenzoate hydroxylase, we show that in the absence of substrate, the large βαβ domain (residues 1–180) and the smaller sheet domain (residues 180–270) separate slightly, and the flavin swings out to a more exposed position to open an aqueous channel from the solvent to the protein interior. Substrate entry occurs by first binding at a surface site and then sliding into the protein interior. In our study of this mutant, the structure of the complex with pyridine nucleotide was obtained. This cofactor binds in an extended conformation at the enzyme surface in a groove that crosses the binding site of FAD. We postulate that for stereospecific reduction, the flavin swings to an out position and NADPH assumes a folded conformation that brings its nicotinamide moiety into close contact with the isoalloxazine moiety of the flavin. This work clearly shows how complex dynamics can play a central role in catalysis by enzymes.

Escherichia coli Soft Metal Ion-translocating ATPases

Life may have first arisen in deep oceanic hydrothermal vents that were rich in metals such as arsenic, lead, copper, and zinc (1). Maintaining suitable intracellular concentrations of essential metals such as copper and zinc while excluding toxic metals such as arsenic, lead, and cadmium was one of the earliest challenges of the first cells. This ancient environmental challenge was a driving force for the evolution of mechanisms for metal ion homeostasis and detoxification. Even today toxic metals enter the ecosphere from geochemical sources. For example, parts of the Midwestern and Northeastern United States have arsenic concentrations that exceed 10 mg/liter, the provisional guideline of the World Health Organization for arsenic in drinking water, or 50 mg/liter, the present United States Environmental Protection Agency recommended maximum. Arsenic in the water supply in southern and western Bangladesh and the adjacent regions of India has triggered a health catastrophe. It is little wonder that in every organism examined there are transport systems that detoxify metal ions by catalyzing extrusion from the cytosol. In this review three families of Escherichia coli transport ATPases that catalyze uptake or confer resistance to ions of the transition metals copper and zinc, the heavy metals cadmium and lead, and the metalloids arsenic and antimony will be described. As a group these pumps will be designated soft metal ion-translocating ATPases or, for convenience, soft metal ATPases, because many ionic species of these elements are chemically soft Lewis acids, as opposed to the hard Lewis acids of Groups I and II elements such as Na and Ca. Hard Lewis acids bind to proteins through relatively weak ionic interactions with hard Lewis bases such as the carboxyl oxygens of glutamate or aspartate residues. In contrast, soft Lewis acids (or simply soft metal ions) form strong bonds with soft Lewis bases such as the thiolates of cysteine residues and the imidazolium nitrogens of histidine residues. These nearly covalent interactions with cysteines and histidines in proteins account for much of the biological properties and toxicity of soft metal ions.

Substrate and Metal Complexes of 3-Deoxy-d-manno-octulosonate-8-phosphate Synthase from Aquifex aeolicus at 1.9-Å Resolution IMPLICATIONS FOR THE CONDENSATION MECHANISM

3-Deoxy-d-manno-octulosonate-8-phosphate synthase (KDO8PS) from the hyperthermophilic bacteriumAquifex 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 Cd2+ 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 tosp 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 siand re sides of C-2PEP, 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-2PEP, thus triggering the beginning of the catalytic cycle.

The α-subunit of the mitochondrial F1 ATPase interacts directly with the assembly factor Atp12p

The Atp12p protein of Saccharomyces cerevisiae is required for the assembly of the F1 component of the mitochondrial F1F0 ATP synthase. In this report, we show that the F1 α‐subunit co‐precipitates and co‐purifies with a tagged form of Atp12p adsorbed to affinity resins. Moreover, sedimentation analysis indicates that in the presence of the F1 α‐subunit, Atp12p behaves as a particle of higher mass than is observed in the absence of the α‐subunit. Yeast two‐hybrid screens confirm the direct association of Atp12p with the α‐subunit and indicate that the binding site for the assembly factor lies in the nucleotide‐binding domain of the α‐subunit, between Asp133 and Leu322. These studies provide the basis for a model of F1 assembly in which Atp12p is released from the α‐subunit in exchange for a β‐subunit to form the interface that contains the non‐catalytic adenine nucleotide‐binding site.

Chaperones of F1-ATPase

Mitochondrial F1-ATPase contains a hexamer of alternating α and β subunits. The assembly of this structure requires two specialized chaperones, Atp11p and Atp12p, that bind transiently to β and α. In the absence of Atp11p and Atp12p, the hexamer is not formed, and α and β precipitate as large insoluble aggregates. An early model for the mechanism of chaperone-mediated F1 assembly (Wang, Z. G., Sheluho, D., Gatti, D. L., and Ackerman, S. H. (2000) EMBO J. 19, 1486–1493) hypothesized that the chaperones themselves look very much like the α and β subunits, and proposed an exchange of Atp11p for α and of Atp12p for β; the driving force for the exchange was expected to be a higher affinity of α and β for each other than for the respective chaperone partners. One important feature of this model was the prediction that as long as Atp11p is bound to β and Atp12p is bound to α, the two F1 subunits cannot interact at either the catalytic site or the noncatalytic site interface. Here we present the structures of Atp11p from Candida glabrata and Atp12p from Paracoccus denitrificans, and we show that some features of the Wang model are correct, namely that binding of the chaperones to α and β prevents further interactions between these F1 subunits. However, Atp11p and Atp12p do not resemble α or β, and it is instead the F1 γ subunit that initiates the release of the chaperones from α and β and their further assembly into the mature complex.

Preliminary X-ray analysis of a new crystal form of the Escherichia coli KDO8P synthase

3-Deoxy-D-manno-octulosonate 8-phosphate (KDO8P) synthase catalyzes the biosynthesis of an essential component of the lipopolysaccharide of all Gram-negative bacteria. The structure and mechanism of KDO8P synthase are being actively studied as this enzyme represents an important target for antibiotic therapy. The structure of the Escherichia coli KDO8P synthase in cubic crystals (space group I23) has recently been determined and the enzyme shown to be a tetramer of identical subunits. However, this information is challenged by biochemical studies, which suggest that the enzyme behaves in solution as a homotrimer. Here, the preparation and preliminary X-ray analysis of monoclinic crystals of KDO8P synthase are reported. The crystals belong to space group P2(1), with unit-cell parameters a approximately 50, b approximately 140, c approximately 74 A, beta approximately 105 degrees. The structure of KDO8P synthase in the monoclinic crystal form was determined by molecular replacement, using as a search model one of the subunits of the enzyme in the cubic crystals. A tetramer of KDO8P synthase with 222 local symmetry is also present in the asymmetric unit of the P2(1) crystals, with a solvent content of 43%. The observation that the same quaternary structure of KDO8P synthase is observed in two different crystal forms belonging to distinct crystal systems (monoclinic and cubic) suggests that a tetramer is the native form of the enzyme.

The Energy Landscape of 3-Deoxy-D-manno-octulosonate 8-Phosphate Synthase †

3-Deoxy-d-manno-octulosonate 8-phosphate (KDO8P) synthase catalyzes the condensation of arabinose 5-phosphate (A5P) and phosphoenolpyruvate (PEP) to form KDO8P, a key precursor in the biosynthesis of the endotoxin of Gram-negative bacteria. Earlier studies have established that the condensation occurs with a syn addition of water to the si side of C2(PEP) and of C3(PEP) to the re side of C1(A5P). Two stepwise mechanisms have been proposed for this reaction. One involves a transient carbanion intermediate, formed by attack of water or a hydroxide ion on C2(PEP). The other involves a transient oxocarbenium zwitterionic intermediate, formed by direct attack of C3(PEP) onto C1(A5P), followed by reaction of water at C2. In both cases, the transient intermediates are expected to converge to a more stable tetrahedral intermediate, which decays into KDO8P and inorganic phosphate. In this study we calculated the potential energy surfaces (PESs) associated with all possible reaction paths in the active site of KDO8PS: the path involving a syn addition of water to the si side of C2(PEP) and of C3(PEP) to the re side of C1(A5P), with the PEP phosphate group deprotonated, has the lowest energy barrier ( approximately 14 kcal/mol) and is strongly exoergonic (reaction energy of -38 kcal/mol). Consistent with the experimental observations, other potential reaction paths, like an anti addition of water to the re side of C2(PEP) or addition of C3(PEP) to the si side of C1(A5P), are associated with much higher barriers. An important new finding of this study is that the lowest energy reaction path does not correspond to either one of the pure stepwise mechanisms proposed formerly but can be described instead as a partially concerted reaction between PEP, A5P, and water. The success in using PESs to reproduce established features of the reaction and to discriminate between different mechanisms suggests that this approach may be of general utility in the study of other enzymatic reactions.