Amino acid substitutions at the subunit interface of dimeric Escherichia coli alkaline phosphatase cause reduced structural stability.

The consequences of amino acid substitutions at the dimer interface for the strength of the interactions between the monomers and for the catalytic function of the dimeric enzyme alkaline phosphatase from Escherichia coli have been investigated. The altered enzymes R10A, R10K, R24A, R24K, T59A, and R10A/R24A, which have amino acid substitutions at the dimer interface, were characterized using kinetic assays, ultracentrifugation, and transverse urea gradient gel electrophoresis. The kinetic data for the wild-type and altered alkaline phosphatases show comparable catalytic behavior with k(cat) values between 51.3 and 69.5 s(-1) and Km values between 14.8 and 26.3 microM. The ultracentrifugation profiles indicate that the wild-type enzyme is more stable than all the interface-modified enzymes. The wild-type enzyme is dimeric in the pH range of pH 4.0 and above, and disassembled at pH 3.5 and below. All the interface-modified enzymes, however, are apparently monomeric at pH 4.0, begin assembly at pH 5.0, and are not fully assembled into the dimeric form until pH 6.0. The results from transverse urea gradient gel electrophoresis show clear and reproducible differences both in the position and the shape of the unfolding patterns; all these modified enzymes are more sensitive to the denaturant and begin to unfold at urea concentrations between 1.0 and 1.5 M; the wild-type enzyme remains in the folded high mobility form beyond 2.5 M urea. Alkaline phosphatase H370A, modified at the active site and not at the dimer interface, resembles the wild-type enzyme both in ultracentrifugation and electrophoresis studies. The results obtained suggest that substitution of a single amino acid at the interface sacrifices not only the integrity of the assembled dimer, but also the stability of the monomer fold, even though the activity of the enzyme at optimal pH remains unaffected and does not appear to depend on interface stability.     

Evolution of homo‐oligomerization of methionine S‐adenosyltransferases is replete with structure–function constrains

Homomers are prevalent in bacterial proteomes, particularly among core metabolic enzymes. Homomerization is often key to function and regulation, and interfaces that facilitate the formation of homomeric enzymes are subject to intense evolutionary change. However, our understanding of the molecular mechanisms that drive evolutionary variation in homomeric complexes is still lacking. How is the diversification of protein interfaces linked to variation in functional regulation and structural integrity of homomeric complexes? To address this question, we studied quaternary structure evolution of bacterial methionine S‐adenosyltransferases (MATs)—dihedral homotetramers formed along a large and conserved dimeric interface harboring two active sites, and a small, recently evolved, interdimeric interface. Here, we show that diversity in the physicochemical properties of small interfaces is directly linked to variability in the kinetic stability of MAT quaternary complexes and in modes of their functional regulation. Specifically, hydrophobic interactions within the small interface of Escherichia coli MAT render the functional homotetramer kinetically stable yet impose severe aggregation constraints on complex assembly. These constraints are alleviated by electrostatic interactions that accelerate dimer‐dimer assembly. In contrast, Neisseria gonorrhoeae MAT adopts a nonfunctional dimeric state due to the low hydrophobicity of its small interface and the high flexibility of its active site loops, which perturbs small interface integrity. Remarkably, in the presence of methionine and ATP, N. gonorrhoeae MAT undergoes substrate‐induced assembly into a functional tetrameric state. We suggest that evolution acts on the interdimeric interfaces of MATs to tailor the regulation of their activity and stability to unique organismal needs.     

Biochemical and mutational analysis of EcoRII functional domains reveals evolutionary links between restriction enzymes

The archetypal Type IIE restriction endonuclease EcoRII is a dimer that has a modular structure. DNA binding studies indicate that the isolated C-terminal domain dimer has an interface that binds a single cognate DNA molecule whereas the N-terminal domain is a monomer that also binds a single copy of cognate DNA. Hence, the full-length EcoRII contains three putative DNA binding interfaces: one at the C-terminal domain dimer and two at each of the N-terminal domains. Mutational analysis indicates that the C-terminal domain shares conserved active site architecture and DNA binding elements with the tetrameric restriction enzyme NgoMIV. Data provided here suggest possible evolutionary relationships between different subfamilies of restriction enzymes.     

pH-Dependent Dimerization of Spider Silk N-Terminal Domain Requires Relocation of a Wedged Tryptophan Side Chain

Formation of spider silk from its constituent proteins—spidroins—involves changes from soluble helical/coil conformations to insoluble β-sheet aggregates. This conversion needs to be regulated to avoid precocious aggregation proximally in the silk gland while still allowing rapid silk assembly in the distal parts. Lowering of pH from about 7 to 6 is apparently important for silk formation. The spidroin N-terminal domain (NT) undergoes stable dimerization and structural changes in this pH region, but the underlying mechanisms are incompletely understood. Here, we determine the NMR and crystal structures of Euprosthenops australis NT mutated in the dimer interface (A72R). Also, the NMR structure of wild‐type (wt) E. australis NT at pH 7.2 and 300 mM sodium chloride was determined. The wt NT and A72R structures are monomers and virtually identical, but they differ from the subunit structure of dimeric wt NT mainly by having a tryptophan (W10) buried between helix 1 and helix 3, while W10 is surface exposed in the dimer. Wedging of the W10 side chain in monomeric NT tilts helix 3 approximately 5–6 Å into a position that is incompatible with that of the observed dimer structure. The structural differences between monomeric and dimeric NT domains explain the tryptophan fluorescence patterns of NT at pH 7 and pH 6 and indicate that the biological function of NT depends on conversion between the two conformations.     

Biochemical, mutational and in silico structural evidence for a functional dimeric form of the ornithine decarboxylase from Entamoeba histolytica

Background

Entamoeba histolytica is responsible for causing amoebiasis. Polyamine biosynthesis pathway enzymes are potential drug targets in parasitic protozoan diseases. The first and rate-limiting step of this pathway is catalyzed by ornithine decarboxylase (ODC). ODC enzyme functions as an obligate dimer. However, partially purified ODC from E. histolytica (EhODC) is reported to exist in a pentameric state.

Methodology and Results

In present study, the oligomeric state of EhODC was re-investigated. The enzyme was over-expressed in Escherichia coli and purified. Pure protein was used for determination of secondary structure content using circular dichroism spectroscopy. The percentages of α-helix, β-sheets and random coils in EhODC were estimated to be 39%, 25% and 36% respectively. Size-exclusion chromatography and mass spectrophotometry analysis revealed that EhODC enzyme exists in dimeric form. Further, computational model of EhODC dimer was generated. The homodimer contains two separate active sites at the dimer interface with Lys57 and Cys334 residues of opposite monomers contributing to each active site. Molecular dynamic simulations were performed and the dimeric structure was found to be very stable with RMSD value ∼0.327 nm. To gain insight into the functional role, the interface residues critical for dimerization and active site formation were identified and mutated. Mutation of Lys57Ala or Cys334Ala completely abolished enzyme activity. Interestingly, partial restoration of the enzyme activity was observed when inactive Lys57Ala and Cys334Ala mutants were mixed confirming that the dimer is the active form. Furthermore, Gly361Tyr and Lys157Ala mutations at the dimer interface were found to abolish the enzyme activity and destabilize the dimer.

Conclusion

To our knowledge, this is the first report which demonstrates that EhODC is functional in the dimeric form. These findings and availability of 3D structure model of EhODC dimer opens up possibilities for alternate enzyme inhibition strategies by targeting the dimer disruption.     

Structural insights into RNA-dependent eukaryal and archaeal selenocysteine formation

The micronutrient selenium is present in proteins as selenocysteine (Sec). In eukaryotes and archaea, Sec is formed in a tRNA-dependent conversion of O-phosphoserine (Sep) by O-phosphoseryl-tRNA:selenocysteinyl-tRNA synthase (SepSecS). Here, we present the crystal structure of Methanococcus maripaludis SepSecS complexed with PLP at 2.5 Å resolution. SepSecS, a member of the Fold Type I PLP enzyme family, forms an (α₂)₂ homotetramer through its N-terminal extension. The active site lies on the dimer interface with each monomer contributing essential residues. In contrast to other Fold Type I PLP enzymes, Asn247 in SepSecS replaces the conserved Asp in binding the pyridinium nitrogen of PLP. A structural comparison with Escherichia coli selenocysteine lyase allowed construction of a model of Sep binding to the SepSecS catalytic site. Mutations of three conserved active site arginines (Arg72, Arg94, Arg307), protruding from the neighboring subunit, led to loss of in vivo and in vitro activity. The lack of active site cysteines demonstrates that a perselenide is not involved in SepSecS-catalyzed Sec formation; instead, the conserved arginines may facilitate the selenation reaction. Structural phylogeny shows that SepSecS evolved early in the history of PLP enzymes, and indicates that tRNA-dependent Sec formation is a primordial process.     

Crystal structure and assembly of the functional Nanoarchaeum equitans tRNA splicing endonuclease

The RNA splicing and processing endonuclease from Nanoarchaeum equitans (NEQ) belongs to the recently identified (αβ)₂ family of splicing endonucleases that require two different subunits for splicing activity. N. equitans splicing endonuclease comprises the catalytic subunit (NEQ205) and the structural subunit (NEQ261). Here, we report the crystal structure of the functional NEQ enzyme at 2.1 Å containing both subunits, as well as that of the NEQ261 subunit alone at 2.2 Å. The functional enzyme resembles previously known α₂ and α₄ endonucleases but forms a heterotetramer: a dimer of two heterodimers of the catalytic subunit (NEQ205) and the structural subunit (NEQ261). Surprisingly, NEQ261 alone forms a homodimer, similar to the previously known homodimer of the catalytic subunit. The homodimers of isolated subunits are inhibitory to heterodimerization as illustrated by a covalently linked catalytic homodimer that had no RNA cleavage activity upon mixing with the structural subunit. Detailed structural comparison reveals a more favorable hetero- than homodimerization interface, thereby suggesting a possible regulation mechanism of enzyme assembly through available subunits. Finally, the uniquely flexible active site of the NEQ endonuclease provides a possible explanation for its broader substrate specificity.     

Three-dimensional structure of ribulose-1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum at 2.9 A resolution

The three-dimensional structure of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from Rhodospirillum rubrum has been determined at 2.9 Å resolution by X-ray crystallographic methods. The MIR-electron density map was substantially improved by two-fold non-crystallographic symmetry averaging. The polypeptide chains in the dimer were traced using a graphics display system with the help of the BONES option in FRODO. The dimer has approximate dimensions of 50 x 72 x 105 Å. The enzyme subunit is a typical two-domain protein. The smaller, N-terminal domain consists of 137 amino acid residues and forms a central, mixed five-stranded β-sheet with α-helices on both sides of the sheet. The larger C-terminal domain consists of 329 amino acid residues. This domain has an eight-stranded parallel α/β barrel structure as found in triosephosphate isomerase and a number of other functionally non-related proteins. The active site in Rubisco determined by difference Fourier techniques and fitting of active site residues to the electron density map, is located at the carboxy-end of the β-strands in the α/β barrel of the C-terminal domain. There are few domain–domain interactions within the subunit. The interactions at the interface between the two subunits of the dimer are tight and extensive. There are tight contacts between the two C-terminal domains, which build up the core of the molecule. There are also interactions between the N-terminal domain of one subunit and the C-terminal domain of the second subunit, close to the active site.     

Subunit interface dynamics in hexadecameric rubisco

Ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (Rubisco) plays an important role in the global carbon cycle as a hub for biomass. Rubisco catalyzes not only the carboxylation of RuBP with carbon dioxide but also a competing oxygenation reaction of RuBP with a negative impact on photosynthetic yield. The functional active site is built from two large (L) subunits that form a dimer. The octameric core of four L₂ dimers is held at each end by a cluster of four small (S) subunits, forming a hexadecamer. Each large subunit contacts more than one S subunit. These interactions exploit the dynamic flexibility of Rubisco, which we address in this study. Here, we describe seven different types of interfaces of hexadecameric Rubisco. We have analyzed these interfaces with respect to the size of the interface area and the number of polar interactions, including salt bridges and hydrogen bonds in a variety of Rubisco enzymes from different organisms and different kingdoms of life, including the Rubisco-like proteins. We have also performed molecular dynamics simulations of Rubisco from Chlamydomonas reinhardtii and mutants thereof. From our computational analyses, we propose structural checkpoints of the S subunit to ensure the functionality and/or assembly of the Rubisco holoenzyme. These checkpoints appear to fine-tune the dynamics of the enzyme in a way that could influence enzyme performance.     

Structure of the dimeric N-glycosylated form of fungal β-N-acetylhexosaminidase revealed by computer modeling, vibrational spectroscopy, and biochemical studies

Background

Fungal β-N-acetylhexosaminidases catalyze the hydrolysis of chitobiose into its constituent monosaccharides. These enzymes are physiologically important during the life cycle of the fungus for the formation of septa, germ tubes and fruit-bodies. Crystal structures are known for two monomeric bacterial enzymes and the dimeric human lysosomal β-N-acetylhexosaminidase. The fungal β-N-acetylhexosaminidases are robust enzymes commonly used in chemoenzymatic syntheses of oligosaccharides. The enzyme from Aspergillus oryzae was purified and its sequence was determined.

Results

The complete primary structure of the fungal β-N-acetylhexosaminidase from Aspergillus oryzae CCF1066 was used to construct molecular models of the catalytic subunit of the enzyme, the enzyme dimer, and the N-glycosylated dimer. Experimental data were obtained from infrared and Raman spectroscopy, and biochemical studies of the native and deglycosylated enzyme, and are in good agreement with the models. Enzyme deglycosylated under native conditions displays identical kinetic parameters but is significantly less stable in acidic conditions, consistent with model predictions. The molecular model of the deglycosylated enzyme was solvated and a molecular dynamics simulation was run over 20 ns. The molecular model is able to bind the natural substrate – chitobiose with a stable value of binding energy during the molecular dynamics simulation.

Conclusion

Whereas the intracellular bacterial β-N-acetylhexosaminidases are monomeric, the extracellular secreted enzymes of fungi and humans occur as dimers. Dimerization of the fungal β-N-acetylhexosaminidase appears to be a reversible process that is strictly pH dependent. Oligosaccharide moieties may also participate in the dimerization process that might represent a unique feature of the exclusively extracellular enzymes. Deglycosylation had only limited effect on enzyme activity, but it significantly affected enzyme stability in acidic conditions. Dimerization and N-glycosylation are the enzyme's strategy for catalytic subunit stabilization. The disulfide bridge that connects Cys⁴⁴⁸ with Cys⁴⁸³ stabilizes a hinge region in a flexible loop close to the active site, which is an exclusive feature of the fungal enzymes, neither present in bacterial nor mammalian structures. This loop may play the role of a substrate binding site lid, anchored by a disulphide bridge that prevents the substrate binding site from being influenced by the flexible motion of the loop.     

Structures of Glycinamide Ribonucleotide Transformylase (PurN) from Mycobacterium tuberculosis Reveal a Novel Dimer with Relevance to Drug Discovery

Enzymes from the de novo purine biosynthetic pathway have been exploited for the development of anti-cancer drugs, and represent novel targets for anti-bacterial drug development. In Mycobacterium tuberculosis, the cause of tuberculosis, this pathway has been identified as essential for growth and survival. The structure of M. tuberculosis PurN (MtPurN) has been determined in complex with magnesium and iodide at 1.30 Å resolution, and with cofactor analogue, 5-methyltetrahydrofolate (5MTHF) at 2.2 Å resolution. The structure shows a Rossmann-type fold that is very similar to the known structures of the human and E. coli PurN proteins. In contrast, MtPurN forms a dimer that is quite different from that formed by the Escherichia coli PurN, and which suggests a mechanism whereby communication could take place between the two active sites. Differences are seen in two active site loops and in the binding mode of the 5MTHF cofactor analogue between the two MtPurN molecules of the dimer. A binding site for halide ions is found in the dimer interface, and bound magnesium and iodide ions in the active site suggest sites that might be exploited in potential drug discovery strategies.     

Modulation of dimer stability in yeast pyrophosphatase by mutations at the subunit interface and ligand binding to the active site

Yeast (Saccharomyces cerevisiae) pyrophosphatase (Y-PPase) is a tight homodimer with two active sites separated in space from the subunit interface. The present study addresses the effects of mutation of four amino acid residues at the subunit interface on dimer stability and catalytic activity. The W52S variant of Y-PPase is monomeric up to an enzyme concentration of 300 microm, whereas R51S, H87T, and W279S variants produce monomer only in dilute solutions at pH > or = 8.5, as revealed by sedimentation, gel electrophoresis, and activity measurements. Monomeric Y-PPase is considerably more sensitive to the SH reagents N-ethylmaleimide and p-hydroxymercurobenzosulfonate than the dimeric protein. Additionally, replacement of a single cysteine residue (Cys(83)), which is not part of the subunit interface or active site, with Ser resulted in insensitivity of the monomer to SH reagents and stabilization against spontaneous inactivation during storage. Active site ligands (Mg(2+) cofactor, P(i) product, and the PP(i) analog imidodiphosphate) stabilized the W279S dimer versus monomer predominantly by decreasing the rate of dimer to monomer conversion. The monomeric protein exhibited a markedly increased (5-9-fold) Michaelis constant, whereas k(cat) remained virtually unchanged, compared with dimer. These results indicate that dimerization of Y-PPase improves its substrate binding performance and, conversely, that active site adjustment through cofactor, product, or substrate binding strengthens intersubunit interactions. Both effects appear to be mediated by a conformational change involving the C-terminal segment that generally shields the Cys(83) residue in the dimer.     

Structural analyses of a purine biosynthetic enzyme from Mycobacterium tuberculosis reveal a novel bound nucleotide

Enzymes of the de novo purine biosynthetic pathway have been identified as essential for the growth and survival of Mycobacterium tuberculosis and thus have potential for the development of anti-tuberculosis drugs. The final two steps of this pathway are carried out by the bifunctional enzyme 5-aminoimidazole-4-carboxamide ribonucleotide transformylase/inosine monophosphate cyclohydrolase (ATIC), also known as PurH. This enzyme has already been the target of anti-cancer drug development. We have determined the crystal structures of the M. tuberculosis ATIC (Rv0957) both with and without the substrate 5-aminoimidazole-4-carboxamide ribonucleotide, at resolutions of 2.5 and 2.2 Å, respectively. As for other ATIC enzymes, the protein is folded into two domains, the N-terminal domain (residues 1–212) containing the cyclohydrolase active site and the C-terminal domain (residues 222–523) containing the formyltransferase active site. An adventitiously bound nucleotide was found in the cyclohydrolase active site in both structures and was identified by NMR and mass spectral analysis as a novel 5-formyl derivative of an earlier intermediate in the biosynthetic pathway 4-carboxy-5-aminoimidazole ribonucleotide. This result and other studies suggest that this novel nucleotide is a cyclohydrolase inhibitor. The dimer formed by M. tuberculosis ATIC is different from those seen for human and avian ATICs, but it has a similar ∼50-Å separation of the two active sites of the bifunctional enzyme. Evidence in M. tuberculosis ATIC for reactivity of half-the-sites in the cyclohydrolase domains can be attributed to ligand-induced movements that propagate across the dimer interface and may be a common feature of ATIC enzymes.     

Perturbation of the dimer interface of triosephosphate isomerase and its effect on Trypanosoma cruzi

Background

Chagas disease affects around 18 million people in the American continent. Unfortunately, there is no satisfactory treatment for the disease. The drugs currently used are not specific and exert serious toxic effects. Thus, there is an urgent need for drugs that are effective. Looking for molecules to eliminate the parasite, we have targeted a central enzyme of the glycolytic pathway: triosephosphate isomerase (TIM). The homodimeric enzyme is catalytically active only as a dimer. Because there are significant differences in the interface of the enzymes from the parasite and humans, we searched for small molecules that specifically disrupt contact between the two subunits of the enzyme from Trypanosoma cruzi but not those of TIM from Homo sapiens (HTIM), and tested if they kill the parasite.

Methodology/Principal Findings

Dithiodianiline (DTDA) at nanomolar concentrations completely inactivates recombinant TIM of T. cruzi (TcTIM). It also inactivated HTIM, but at concentrations around 400 times higher. DTDA was also tested on four TcTIM mutants with each of its four cysteines replaced with either valine or alanine. The sensitivity of the mutants to DTDA was markedly similar to that of the wild type. The crystal structure of the TcTIM soaked in DTDA at 2.15 Å resolution, and the data on the mutants showed that inactivation resulted from alterations of the dimer interface. DTDA also prevented the growth of Escherichia coli cells transformed with TcTIM, had no effect on normal E. coli, and also killed T. cruzi epimastigotes in culture.

Conclusions/Significance

By targeting on the dimer interface of oligomeric enzymes from parasites, it is possible to discover small molecules that selectively thwart the life of the parasite. Also, the conformational changes that DTDA induces in the dimer interface of the trypanosomal enzyme are unique and identify a region of the interface that could be targeted for drug discovery.     

X-ray structures of Aerococcus viridans lactate oxidase and its complex with D-lactate at pH 4.5 show an alpha-hydroxyacid oxidation mechanism

l-Lactate oxidase (LOX) belongs to a family of flavin mononucleotide (FMN)-dependent α-hydroxy acid-oxidizing enzymes. Previously, the crystal structure of LOX (pH 8.0) from Aerococcus viridans was solved, revealing that the active site residues are located around the FMN. Here, we solved the crystal structures of the same enzyme at pH 4.5 and its complex with d-lactate at pH 4.5, in an attempt to analyze the intermediate steps. In the complex structure, the d-lactate resides in the substrate-binding site, but interestingly, an active site base, His265, flips far away from the d-lactate, as compared with its conformation in the unbound state at pH 8.0. This movement probably results from the protonation of His265 during the crystallization at pH 4.5, because the same flip is observed in the structure of the unbound state at pH 4.5. Thus, the present structure appears to mimic an intermediate after His265 abstracts a proton from the substrate. The flip of His265 triggers a large structural rearrangement, creating a new hydrogen bonding network between His265-Asp174-Lys221 and, furthermore, brings molecular oxygen in between d-lactate and His265. This mimic of the ternary complex intermediate enzyme-substrate-O₂ could explain the reductive half-reaction mechanism to release pyruvate through hydride transfer. In the mechanism of the subsequent oxidative half-reaction, His265 flips back, pushing molecular oxygen into the substrate-binding site as the second substrate, and the reverse reaction takes place to produce hydrogen peroxide. During the reaction, the flip-flop action of His265 has a dual role as an active base/acid to define the major chemical steps. Our proposed reaction mechanism appears to be a common mechanistic strategy for this family of enzymes.     

Structure of a complex between yeast hexokinase A and glucose: II. Detailed comparisons of conformation and active site configuration with the native hexokinase B monomer and dimer

Detailed comparison of the refined crystal structures of the hexokinase A: glucose complex (HKA · G) and native hexokinase B shows that, in addition to the 12 ° rotation of one lobe of the enzyme relative to the other as described previously (Bennett & Steitz, 1978) there are small systematic differences in the conformation of the polypeptide backbones of the two structures adjacent to the glucose binding site and crystal packing contacts. In the HKA · G complex, the cleft between the two lobes of the hexokinase molecule is narrowed, substantially reducing the accessibility of the active site to solvent. The HKA · G structure suggests specific contacts with a bound glucose molecule that cannot form in the more open native structure. The closed conformation of the HKA · G complex can be formed by either subunit in the heterologous dimer configuration of hexokinase B (Anderson et al. 1974); new or different interactions between subunits, or with ligands bound to the intersubunit ATP site, may be made when the upper subunit of the dimer is in the closed conformation and may contribute to the cooperative interactions observed in the crystalline dimer and in solution.     

The crystal structure of the catalytic domain of the site-specific recombination enzyme gamma delta resolvase at 2.7 A resolution

The crystal structure of the catalytic domain of the site-specific recombination enzyme gamma delta resolvase has been determined at 2.7 A resolution. Its first 120 amino acids form a central five-stranded, beta-pleated sheet surrounded by five alpha helices. In one of the four dyad-related dimers, the two active site Ser-10 residues are 19 A apart, perhaps close enough to contact and become covalently linked to the DNA at the recombination site. This dimer also forms the only closely packed tetramer found in the crystal. The subunit interface at a second dyad-related dimer is more extensive and more highly conserved among the homologous recombinases; however, its active site Ser-10 residues are more than 30 A apart. Side chains, identified by mutations that eliminate catalysis but not DNA binding, are located on the subunit surface near the active site serine and at the interface between a third dyad-related pair of subunits of the tetramer.     

pH-dependent kinetics of NO release from E. coli bd-I and bd-II oxidase reveals involvement of Asp/Glu58B

Escherichia coli contains two cytochrome bd oxidases, bd-I and bd-II. The structure of both enzymes is highly similar, but they exhibit subtle differences such as the accessibility of the active site through a putative proton channel. Here, we demonstrate that the duroquinol:dioxygen oxidoreductase activity of bd-I increased with alkaline pH, whereas bd-II showed a broad activity maximum around pH 7. Likewise, the pH dependence of NO release from the reduced active site, an essential property of bd oxidases, differed between the two oxidases as detected by UV/vis spectroscopy. Both findings may be attributed to differences in the proton channel leading to the active site heme d. The channel comprises a titratable residue (Asp58^B in bd-I and Glu58^B in bd-II). Conservative mutations at this position drastically altered NO release demonstrating its contribution to the process.     

Interactions between the catalytic centers and subunit interface of triosephosphate isomerase probed by refolding, active site modification, and subunit exchange.

The effects of unfolding, refolding, and hybridization of triosephosphate isomerase (TPI) subunits from different species and subunits which have been specifically modified at the active site have been examined. These effects have been evaluated in terms of changes in catalytic parameters, CD spectra, and susceptibility to denaturation. Dissociation followed by reassociation yields an active dimer but with increased Km, reduced kcat, and increased susceptibility to inactivation and unfolding in denaturants. These data suggest that while the general structure of the refolded dimer is similar to the native enzyme, its complete original structure is not restored. Covalent reaction of the active site Glu165 with the substrate analogue 3-chloroacetol phosphate (CAP) results in dimers with increased susceptibility to unfolding and inactivation by denaturants (i.e. the rates of inactivation and unfolding are (TPICAP)2 greater than (TPI-TPICAP) greater than (TPI)2). These data point to the interactions between the catalytic center and the subunit interface. Subunits of TPI from different species, in spite of structural differences at the subunit interface, hybridized to active heterodimers. Subunit hybridization was random among monomers from different mammals, preferential between yeast and mammalian or avian monomers. Hybridization did not occur between avian and mammalian monomers under these conditions. These data provide information on the elements in the interface of the dimer and the relationship of the catalytic center with the subunit interface.     

Unraveling the <Emphasis Type="Italic">Helicobacter pylori</Emphasis> UreG zinc binding site using X-ray absorption spectroscopy (XAS) and structural modeling

Abstract  

The pathogenicity of Helicobacter pylori depends on the activity of urease for pH modification. Urease activity requires assembly of a dinickel active site that is facilitated in part by GTP hydrolysis by UreG. The proper functioning of Helicobacter pylori UreG (HpUreG) is dependent on Zn(II) binding and dimerization. X-ray absorption spectroscopy and structural modeling were used to elucidate the structure of the Zn(II) site in HpUreG. These studies independently indicated a site at the dimer interface that has trigonal bipyramidal geometry and is composed of two axial cysteines at 2.29(2) ?, two equatorial histidines at 1.99(1) ?, and a solvent-accessible coordination site. The final model for the Zn(II) site structure was determined by refining multiple-scattering extended X-ray absorption fine structure fits using the geometry predicted by homology modeling and ab initio calculations.