Coupled enzymatic production of sulfite, thiosulfate, and hydrogen sulfide from sulfur: purification and properties of a sulfur oxygenase reductase from the facultatively anaerobic archaebacterium Desulfurolobus ambivalens.

From aerobically grown cells of the extremely thermophilic, facultatively anaerobic chemolithoautotrophic archaebacterium Desulfurolobus ambivalens (DSM 3772), a soluble oxygenase reductase (SOR) was purified which was not detectable in anaerobically grown cells. In the presence of oxygen but not under a hydrogen atmosphere, the enzyme simultaneously produced sulfite, thiosulfate, and hydrogen sulfide from sulfur. Nonenzymatic control experiments showed that thiosulfate was produced mainly in a chemical reaction between sulfite and sulfur. The maximum specific activity of the purified SOR in sulfite production was 10.6 mumol/mg of protein at pH 7.4 and 85 degrees C. The ratio of sulfite to hydrogen sulfide production was 5:4 in the presence of zinc ions. The temperature range of enzyme activity was 50 to 108 degrees C, with a maximum at 85 degrees C. The molecular mass of the native SOR was 550 kilodaltons, determined by gel filtration. It consisted of identical subunits with an apparent molecular mass of 40 kilodaltons in sodium dodecyl sulfate-gel electrophoresis. The particle diameter in electron micrographs was 15 /+- 1.5 nm. The enzyme activity was inhibited by the thiol-binding reagents p-chloromercuribenzoic acid, N-ethyl maleimide, and 2-iodoacetic acid and by flavin adenine dinucleotide, Fe3+, and Fe2+. It was not affected by CN-, N3-, or reduced glutathione.     

Crystal structure of muconate lactonizing enzyme at 6.5 A resolution

We have obtained crystals of Pseudomonas putida muconate lactonizing enzyme. They diffract to better than 2.4 A resolution and have two monomers in the asymmetric unit, related by a non-crystallographic 2-fold axis. The cell dimensions are 139.3 A X 139.3 A X 84.1 A, and the space group is I4. The electron density map at 6.5 A resolution shows that the enzyme is an octamer with D4 symmetry.     

Refinement of the influenza virus hemagglutinin by simulated annealing

We have applied the method of simulated annealing to the refinement of the 3 A resolution crystal structure of the influenza virus hemagglutinin glycoprotein, using the program X-PLOR. Two different methods were introduced into X-PLOR to treat the non-crystallographic symmetry present in this and in other crystal structures. In the first, only the unique protomer atoms are refined; by application of the non-crystallographic symmetry operators to the protomer atoms, the X-ray structure factor derivatives are effectively averaged, and a non-bonded energy term models the interactions of the protomer with its neighbors in the oligomer without explicit refinement of the other protomers in the crystallographic asymmetric unit. In the second method, the entire asymmetric unit is refined, but an effective energy term is added to the empirical energy that restrains symmetry-related atomic positions to their average values after least-squares superposition. Several other modifications and additions were made to previously published X-PLOR protocols, including weighting of the X-ray terms, maintenance of the temperature of the molecular dynamics simulation, treatment of charged groups, changes in the values of certain empirical energy parameters, and the use of N-linked carbohydrate empirical energy parameters. The hemagglutinin refinement proceeded in several stages. An initial round of simulated annealing of the monomer was followed by rigid-body refinement of the 3-fold non-crystallographic symmetry axis position and a second round of monomer refinement. A third round was performed on the trimer using non-crystallographic symmetry restraints in all regions except those in lattice contacts showing obvious derivations from 3-fold symmetry. The refinement was completed with several rounds of conventional positional and isotropic temperature factor refinement needed to correct bad model geometry introduced by high-temperature molecular dynamics in regions of weak electron density. This structure was then used as the basis for refinement of three crystallographically isomorphous hemagglutinin structures, including complexes with the influenza virus receptor, sialic acid. Model geometry comparable to well-refined high-resolution structures was obtained with relatively little manual intervention, demonstrating the ability of simulated annealing refinement to produce highly idealized structures at moderate resolution.     

Structure of enoate reductase from a Clostridium tyrobutyricum (C. spec. La1)

Enoate reductase from Clostridium tyrobutyricum was purified by a rapid novel procedure. Chromatography on DEAE-Sepharose and on hydroxyapatite resulted in a high yield of about 90% pure enzyme in less than 10 h. A purity greater than 98% could be obtained by additional chromatography on Sephacryl S-300. The enzyme sediments in the analytical ultracentrifuge as a single, symmetrical boundary with a velocity of S(0)20,w = 24.9 S. Equilibrium ultracentrifugation yielded a molecular mass of 940 000 +/- 20 000 Da. The enzyme contains one type of subunit as shown by dodecyl sulfate electrophoresis and partial sequence determination. A subunit molecular mass of about 73 000 Da was established by dodecyl sulfate electrophoresis and by sedimentation equilibrium analysis in guanidine hydrochloride. In addition to FAD, iron and labile sulfur, the enzyme purified by the new method showed approximately 0.7 mol of FMN per mol of subunit. A dissociation product sedimenting at a velocity of S(0)20,w = 9.8 S can be obtained by various experimental protocols. The fragment was obtained in pure form by gel permeation chromatography. The molecular mass was 230 000 +/- 10 000 Da as shown by sedimentation equilibrium analysis. Thus it appears that the dissociation product is a trimer of the 73 000-Da subunit. The formation of the 10-S fragment by dissociation of the native enzyme is accompanied by the loss of most of the FMN, whereas the FAD content is not changed. The fragment catalysed the reduction of acetylpyridine adenine dinucleotide by NADH. However, enoate reductase activity with NADH or methylviologen as cosubstrate was low. Electron micrographs of negatively stained enoate reductase show trigonal symmetry. The data suggest that enoate reductase is a dodecamer (tetramer of trimers) with tetrahedral symmetry.     

Crystallization of an NADP+-dependent malic enzyme from rat liver

Crystals of a tetrameric NADP+-dependent malic enzyme from rat liver have been grown in the presence of NADP+ using the hanging-drop method of vapour diffusion with ammonium sulphate as the precipitant. Measurement of the crystal density and calculation of the values of Vm for different numbers of polypeptide chains in the unit cell indicate that the asymmetric unit of the crystal contains a complete tetramer, allowing the application of non-crystallographic symmetry to the determination of the molecular structure of this enzyme. This structure would provide only the second example for an enzyme involved in oxidative decarboxylation, the other being 6-phosphogluconate dehydrogenase. In addition, then, to providing an insight into the structure-function relationship in malic enzyme, the successful structure determination would permit valuable comparisons to be made between these two and other enzymes with this catalytic activity.     

Preliminary crystallographic data for the thiamin diphosphate-dependent enzyme pyruvate decarboxylase from brewers' yeast

Single crystals of the thiamin diphosphate (the vitamin B1 coenzyme)-dependent enzyme pyruvate decarboxylase (EC 4.1.1.1) from brewers' yeast have been grown using polyethylene glycol as a precipitating agent. Crystals of the homotetrameric version alpha 4 of the holoenzyme are triclinic, space group P1, with cell constants a = 81.0, b = 82.4, c = 116.6 A, alpha = 69.5 beta = 72.6, gamma = 62.4 degrees. The crystals are reasonably stable in a rotating anode x-ray beam and diffract to at least 2.5 A resolution. The Vm value of 2.55 A/dalton is consistent with a unit cell containing four subunits with mass of approximately 60 kDa each. Rotation function results with native data indicate strong non-crystallographic 222 symmetry relating the four identical subunits, thus density averaging methods are likely to play a role in the structure determination.     

Multi-isomorphous replacement phasing of the earthworm fibrinolytic enzyme component A from Eisenia fetida

Thrombosis is one of the most widely occurring diseases in modern life, which often causes disability and death. Fibrinolytic enzymes degrade fibrin, the major protein component of blood clots, and eventually lead to thrombolysis. Medications using fibrinolytic enzymes are the most effective methods used in the treatment of thrombosis. A variety of fibrinolytic enzymes, such as tPA, uPA, and streptokinase, have been extensively studied and used as thrombolytic agents in clinic. However, thes…     

Using cryoEM Reconstruction and Phase Extension to Determine Crystal Structure of Bacteriophage ϕ6 Major Capsid Protein

Bacteriophage ?6 is a double-stranded RNA virus that has been extensively studied as a model organism. Here we describe structure determination of ?6 major capsid protein P1. The protein crystallized in base centered orthorhombic space group C222₁. Matthews’s coefficient indicated that the crystals contain from four to seven P1 subunits in the crystallographic asymmetric unit. The self-rotation function had shown presence of fivefold axes of non-crystallographic symmetry in the crystals. Thus, electron density map corresponding to a P1 pentamer was excised from a previously determined cryoEM reconstruction of the ?6 procapsid at 7 Å resolution and used as a model for molecular replacement. The phases for reflections at higher than 7 Å resolution were obtained by phase extension employing the fivefold non-crystallographic symmetry present in the crystal. The averaged 3.6 Å-resolution electron density map was of sufficient quality to allow model building.     

Finding non-crystallographic symmetry in density maps of macromolecular structures

The internal symmetry of a macromolecule is both an important aspect of its function and a useful feature in obtaining a structure by X-ray crystallography and other techniques. A method is presented for finding internal symmetry and other non-crystallographic symmetry in a structure based on patterns of density in a density map for that structure. Regions in map that are similar are identified by cutting out a sphere of density from a region that has high local variation and using an FFT-based correlation search to find other regions that match. The relationships among correlated regions are then refined to maximize their correlations and are found to accurately represent non-crystallographic symmetry in the map.     

Crystal structure studies on sulfur oxygenase reductase from Acidianus tengchongensis

Sulfur oxygenase reductase (SOR) simultaneously catalyzes oxidation and reduction of elemental sulfur to produce sulfite, thiosulfate, and sulfide in the presence of molecular oxygen. In this study, crystal structures of wild type and mutants of SOR from Acidianus tengchongensis (SOR-AT) in two different crystal forms were determined and it was observed that 24 identical SOR monomers form a hollow sphere. Within the icosatetramer sphere, the tetramer and trimer channels were proposed as the paths for the substrate and products, respectively. Moreover, a comparison of SOR-AT with SOR-AA (SOR from Acidianus ambivalens) structures showed that significant differences existed at the active site. Firstly, Cys31 is not persulfurated in SOR-AT structures. Secondly, the iron atom is five-coordinated rather than six-coordinated, since one of the water molecules ligated to the iron atom in the SOR-AA structure is lost. Consequently, the binding sites of substrates and a hypothetical catalytic process of SOR were proposed.     

First characterisation of the active oligomer form of sulfur oxygenase reductase from the bacterium Aquifex aeolicus

Sulfur oxygenase reductase (SOR) enzyme is responsible for the initial oxidation step of elemental sulfur in archaea. Curiously, Aquifex aeolicus, a hyperthermophilic, chemolithoautotrophic and microaerophilic bacterium, has the SOR-encoding gene in its genome. We showed, for the first time the presence of the SOR enzyme in A. aeolicus, its gene was cloned and recombinantly expressed in Escherichia coli and the protein was purified and characterised. It is a 16 homo-oligomer of approximately 600 kDa that contains iron atoms indispensable for the enzyme activity. The optimal temperature of SOR activity is 80°C and it is inactive at 20°C. Studies of the factors involved in getting the fully active molecule at high temperature show clearly that (1) incubation at high temperature induces more homogeneous form of the enzyme, (2) conformational changes observed at high temperature are required to get the fully active molecule and (3) acquisition of an active conformation induced by the temperature seems to be more important than the subunit number. Differences between A. aeolicus SOR and the archaea SORs are described.     

Low resolution crystal structure of muconolactone isomerase. A decamer with a 5-fold symmetry axis

Muconolactone isomerase from Pseudomonas putida crystallizes from sodium sulfate solution in space group P2(1) (a = 65.84 A, b = 105.70 A, c = 77.20 A, beta = 90.5 degrees) with ten 11,000 Mr subunits per asymmetric unit. The 7 A resolution crystal structure was solved by single isomorphous replacement followed by 10-fold symmetry averaging. The decameric enzyme has an uncommon non-crystallographic 5-fold symmetry axis and a large cavity in its center.     

Preliminary crystallographic studies of Limulus polyphemus hemocyanin subunits IIIa,IIIb and IV

Crystals of Limulus hemocyanin subunits IIIa, IIIb and IV are suitable for X-ray diffraction analysis. The three-dimensional structure of subunit IV is determined by molecular replacement and non-crystallographic symmetry averaging methods. A tentative model of subunit IIIa is obtained from a partial data set. Both structures, similar to subunit II, could provide primary structure segments suitable for oligonucleotide probe synthesis.     

Purification and properties of the sulfur oxygenase/reductase from the acidothermophilic archaeon, <Emphasis Type="Italic">Acidianus </Emphasis>strain S5

The sulfur oxygenase/reductase (SOR) of Acidianus strain S5 was purified and characterized after expressing the SOR gene in a recombinant strain of Escherichia coli. The N-terminal sequence of the purified SOR protein was the same as the deduced amino acid sequence from previously cloned SOR genes. Enzymatic studies indicated that the SOR catalyzed the conversion of elemental sulfur (S(o)) to sulfite, thiosulfate, and sulfide. The optimal pH and temperature were 5.0 and 70 degrees C, respectively. Comparison of this SOR and that of A. ambivalens revealed several differences between these two SORs. The most striking difference is that the SOR of Acidianus S5 had maximal activity at acidic pH. By application of anti-SOR serum and the Western blot technique, it was found that SOR proteins existed in A. brierleyi and in Acidianus S5 cells cultivated with thiosulfate as the sole energy source, indicating that SOR may also play a role in thiosulfate metabolism.     

Three-dimensional structure of Cu,Zn-superoxide dismutase from spinach at 2.0 A resolution

The three-dimensional structure of Cu,Zn-superoxide dismutase from spinach leaves has been determined by X-ray crystal structure analysis. The atomic coordinates were refined at 2.0 A resolution using the Hendrickson and Konnert program for stereochemically restrained refinement against structure factors, which allowed the use of non-crystallographic symmetry. The crystallographic residual error for the refined model was 24.9%, with a root mean square deviation of 0.03 A from the ideal bond length and an average atomic temperature factor of 9.6 A. A dimeric molecule of the enzyme is comprised of two identical subunits related by a non-crystallographic 2-fold axis. Each subunit of 154 amino acid residues is composed primarily of eight anti-parallel beta-strands that form a flattened cylinder, plus three external loops. The main-chain hydrogen bonds primarily link the beta-strands. The overall structure of this enzyme is quite similar to that of the bovine dismutase except for some parts. The single disulfide bridge (Cys57-Cys146) and the salt bridge (Arg79-Asp101) may stabilize the loop regions of the structure. The Cu2+ and Zn2+ ions in the active site lie 6.1 A apart at the bottom of the long channel. The Cu2+ ligands (ND1 of His-46, and NE2 of His-48, -63, and -120) show an uneven tetrahedral distortion from a square plane. The Zn2+ ligands (ND1 of His-63, -71, and -80 and OD1 of Asp-83) show an almost tetrahedral geometry. The imidazole ring of His-63 forms a bridge between the Cu2+ and Zn2+ ions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Crystal structure of the pyridoxal 5'-phosphate dependent L-methionine gamma-lyase from Pseudomonas putida

L-Methionine gamma-lyase (MGL) catalyzes the pyridoxal 5'-phosphate (PLP) dependent alpha,gamma-elimination of L-methionine. We have determined two crystal structures of MGL from Pseudomonas putida using MAD (multiwavelength anomalous diffraction) and molecular replacement methods. The structures have been refined to an R-factor of 21.1% at 2.0 and 1.7 A resolution using synchrotron radiation diffraction data. A homotetramer with 222 symmetry is built up by non-crystallographic symmetry. Two monomers associate to build the active dimer. The spatial fold of subunits, with three functionally distinct domains and their quarternary arrangement, is similar to those of L-cystathionine beta-lyase and L-cystathionine gamma-synthase from Escherichia coli.     

Structure determination of the constitutive 20S proteasome from bovine liver at 2.75 A resolution

The crystal structure of the 20S proteasome from bovine liver was determined by the molecular replacement method using the structure of the 20S proteasome from the yeast Sacccharomyces cerevisiae. The initial phases were refined by density modification coupled with non-crystallographic symmetry averaging. The structural model was refined with the program CNS. The final R-factor and R(free) were 0.25 and 0.29, respectively. The constitutive proteasome without any contamination by the immunoproteasome was identified in the crystal structure.     

Roles of sulfite oxidoreductase and sulfite reductase in improving desulfurization by Rhodococcus erythropolis

Rhodococcus erythropolis has been widely studied for desulfurization. However, activity levels required for commercial application have not been achieved. A major limitation of the current work in biodesulfurization is inadequate information regarding sulfur metabolism generally, and in particular the metabolism of the sulfur obtained from dibenzothiophene (DBT) metabolism via the 4S pathway. In this work, we have investigated the possible routes taken by the sulfur from DBT to convert into biomass or other metabolites. We propose two alternate hypotheses. In the first, we hypothesize that the cell can convert via sulfite reductase (SR) the sulfite from the metabolism of DBT into sulfide that can be assimilated into biomass. However, in the process, it may convert any excess sulfite into extracellular sulfate via sulfite oxidoreductase (SOR) to avoid the toxic effects of sulfite. In the second, we speculate that the cell cannot assimilate the sulfite directly into biomass via SR. It must first use SOR to produce extracellular sulfate, and then recapture that sulfate into biomass via SR. Thus, either way, we propose that SOR and SR activities, in addition to dsz genes and cofactors, may be critical in increasing desulfurization levels significantly. In particular, we suggest that the simultaneous increase in SOR activity and decrease in SR activity can enable increased desulfurization activity.     

Crystal structure of the non-regulatory A(4 )isoform of spinach chloroplast glyceraldehyde-3-phosphate dehydrogenase complexed with NADP

Here, we report the first crystal structure of a photosynthetic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) complexed with NADP. The enzyme, purified from spinach chloroplasts, is constituted of a single type of subunit (A) arranged in homotetramers. It shows non-regulated NADP-dependent and NAD-dependent activities, with a preference for NADP. The structure has been solved to 3.0 A resolution by molecular replacement. The crystals belong to space group C222 with three monomers in the asymmetric unit. One of the three monomers generates a tetramer using the space group 222 point symmetry and a very similar tetramer is generated by the other two monomers, related by a non-crystallographic symmetry, using a crystallographic 2-fold axis.The protein reveals a large structural homology with known GAPDHs both in the cofactor-binding domain and in regions of the catalytic domain. Like all other GAPDHs investigated so far, the A(4)-GAPDH belongs to the Rossmann fold family of dehydrogenases. However, unlike most dehydrogenases of this family, the adenosine 2'-phosphate group of NADP does not form a salt-bridge with any positively charged residue in its surroundings, being instead set in place by hydrogen bonds with a threonine residue belonging to the Rossmann fold and a serine residue located in the S-loop of a symmetry-related monomer. While increasing our knowledge of an important photosynthetic enzyme, these results contribute to a general understanding of NADP versus NAD recognition in pyridine nucleotide-dependent enzymes.Although the overall structure of A(4)-GAPDH is similar to that of the cytosolic GAPDH from bacteria and eukaryotes, the chloroplast tetramer is peculiar, in that it can actually be considered a dimer of dimers, since monomers are bound in pairs by a disulphide bridge formed across Cys200 residues. This bridge is not found in other cytosolic or chloroplast GAPDHs from animals, bacteria, or plants other than spinach.     

Three-dimensional model of yeast RNA polymerase I determined by electron microscopy of two-dimensional crystals.

Two-dimensional crystals of yeast RNA polymerase I dimers were obtained upon interaction with positively charged lipid layers. A three-dimensional surface model of the enzyme was determined by analyzing tilted crystalline areas and by taking advantage of the non-crystallographic internal symmetry of the dimer to correct for the missing viewing directions. The structure shows, at approximately 3 nm resolution, an irregularly shaped molecule 11 nm x 11 nm x 15 nm in size characterized by a 3 nm wide and 10 nm long groove which constitutes a putative DNA binding site. The overall structure is similar to the Escherichia coli holo enzyme and the yeast RNA polymerase II delta 4/7 structures. The most remarkable structural feature is a finger-shaped stalk which partially occludes the entrance of the groove and forms a 2.5 nm wide channel. We discuss the possible location of the catalytic centre and of the carboxy-terminal region of the beta-like subunit in the channel. The interference of different DNA fragments with RNA polymerase dimerization and crystallization indicates the orientation of the template in the putative DNA binding groove.