Thermostabilization of proteins by diglycerol phosphate, a new compatible solute from the hyperthermophile Archaeoglobus fulgidus

Diglycerol phosphate accumulates under salt stress in the archaeon Archaeoglobus fulgidus (L. O. Martins, R. Huber, H. Huber, K. O. Stetter, M. S. da Costa, and H. Santos, Appl. Environ. Microbiol. 63:896-902, 1997). This solute was purified after extraction from the cell biomass. In addition, the optically active and the optically inactive (racemic) forms of the compound were synthesized, and the ability of the solute to act as a protecting agent against heating was tested on several proteins derived from mesophilic or hyperthermophilic sources. Diglycerol phosphate exerted a considerable stabilizing effect against heat inactivation of rabbit muscle lactate dehydrogenase, baker's yeast alcohol dehydrogenase, and Thermococcus litoralis glutamate dehydrogenase. Highly homologous and structurally well-characterized rubredoxins from Desulfovibrio gigas, Desulfovibrio desulfuricans (ATCC 27774), and Clostridium pasteurianum were also examined for their thermal stabilities in the presence or absence of diglycerol phosphate, glycerol, and inorganic phosphate. These proteins showed different intrinsic thermostabilities, with half-lives in the range of 30 to 100 min. Diglycerol phosphate exerted a strong protecting effect, with approximately a fourfold increase in the half-lives for the loss of the visible spectra of D. gigas and C. pasteurianum rubredoxins. In contrast, the stability of D. desulfuricans rubredoxin was not affected. These different behaviors are discussed in the light of the known structural features of rubredoxins. The data show that diglycerol phosphate is a potentially useful protein stabilizer in biotechnological applications.     

Structural determinants of protein stabilization by solutes. The important of the hairpin loop in rubredoxins

Despite their high sequence homology, rubredoxins from Desulfovibrio gigas and D. desulfuricans are stabilized to very different extents by compatible solutes such as diglycerol phosphate, the major osmolyte in the hyperthermophilic archaeon Archaeoglobus fulgidus[Lamosa P, Burke A, Peist R, Huber R, Liu M Y, Silva G, Rodrigues-Pousada C, LeGall J, Maycock C and Santos H (2000) Appl Environ Microbiol66, 1974-1979]. The principal structural difference between these two proteins is the absence of the hairpin loop in the rubredoxin from D. desulfuricans. Therefore, mutants of D. gigas rubredoxin bearing deletions in the loop region were constructed to investigate the importance of this structural feature on protein intrinsic stability, as well as on its capacity to undergo stabilization by compatible solutes. The three-dimensional structure of the mutant bearing the largest deletion, Delta17/29, was determined by 1H-NMR, demonstrating that, despite the drastic deletion, the main structural features were preserved. The dependence of the NH chemical shifts on temperature and solute concentration (diglycerol phosphate or mannosylglycerate) provide evidence of subtle conformational changes induced by the solute. The kinetic stability (as assessed from the absorption decay at 494 nm) of six mutant rubredoxins was determined at 90 degrees C and the stabilizing effect exerted by both solutes was assessed. The extent of protection conferred by each solute was highly dependent on the specific mutant examined: while the half-life for iron release in the wild-type D. gigas rubredoxin increased threefold in the presence of 0.1 M diglycerol phosphate, mutant Delta23/29 was destabilized. This study provides evidence for solute-induced compaction of the protein structure and occurrence of weak, specific interactions with the protein surface. The relevance of these findings to our understanding of the molecular basis for protein stabilization is discussed.     

Phospholipid Composition of Desulfovibrio Species

The phospholipids of Desulfovibrio desulfuricans, Norway strain, D. vulgaris, and D. gigas were examined in relationship to their qualitative and quantitative composition. D. desulfuricans and D. vulgaris exhibited an essentially identical phospholipid composition consisting of phosphatidylethanolamine, phosphatidylglycerol, cardiolipin, and lysophosphatidylserine. Phosphatidylserine (10.9%) was present in D. desulfuricans but was not detected in D. vulgaris. D. gigas was found to contain only two phospholipids, phosphatidylethanolamine (30%) and phosphatidylglycerol (70%). An ornithine-containing lipid was detected in D. gigas which was not present in the other two Desulfovibrio species.     

Immobilization of hydrogenases for biophotolytic hydrogen production; stability and kinetics

Summary Hydrogenases from Clostridium pasteurianum and Desulfovibrio desulfuricans were immobilized on solid supports with retention of 50% activity. The immobilized enzymes were more stable than the free enzymes and were active in the biophotolytic hydrogen production from water.     

Reduction of molybdate by sulfate-reducing bacteria

Molybdate is an essential trace element required by biological systems including the anaerobic sulfate-reducing bacteria (SRB); however, detrimental consequences may occur if molybdate is present in high concentrations in the environment. While molybdate is a structural analog of sulfate and inhibits sulfate respiration of SRB, little information is available concerning the effect of molybdate on pure cultures. We followed the growth of Desulfovibrio gigas ATCC 19364, Desulfovibrio vulgaris Hildenborough, Desulfovibrio desulfuricans DSM 642, and D. desulfuricans DSM 27774 in media containing sub-lethal levels of molybdate and observed a red-brown color in the culture fluid. Spectral analysis of the culture fluid revealed absorption peaks at 467, 395 and 314 nm and this color is proposed to be a molybdate–sulfide complex. Reduction of molybdate with the formation of molybdate disulfide occurs in the periplasm D. gigas and D. desulfuricans DSM 642. From these results we suggest that the occurrence of poorly crystalline Mo-sulfides in black shale may be a result from SRB reduction and selective enrichment of Mo in paleo-seawater.     

The immunological properties of the Clostridium pasteurianum rubredoxin

An antibody to Clostridium pasteurianum rubredoxin was found in goat serum after multiple injections of the protein. This antibody was purified by adsorption and elution from a Sepharose-rubredoxin column. The purified antibody formed a precipitating complex with C. pasteurianum rubredoxin and aporubredoxin, but not with the rubredoxin from Micrococcus aerogenes, Peptostreptococcus elsdenii, and Pseudomonas oleovorans. All rubredoxins tested were adsorbed to Sepharose-antirubredoxin columns indicating that each could form a soluble complex with anti-C. pasteurianum rubredoxin. The relative affinity of the antirubredoxin for the various rubredoxins was demonstrated by its ability to inhibit the rubredoxin-dependent reduction of cytochrome c by NADPH in the presence of NADP-ferredoxin oxidoreductase. These data suggest that there are two antigenic determinants in C. pasteurianum rubredoxin and only one such determinant in the rubredoxin from other organisms which are recognized by anti-C. pasteurianum rubredoxin.     

Comparative study of the thermostabilizing properties of mannosylglycerate and other compatible solutes on model enzymes

The protection of mannosylglycerate, at 0.5 M concentration, against heat inactivation of the model enzyme lactate dehydrogenase (LDH) was compared to that exerted by other compatible solutes, namely, trehalose, ectoine, hydroxyectoine, di- myo-inositol phosphate, diglycerol phosphate, and mannosylglyceramide. Mannosylglycerate and hydroxyectoine were the best stabilizers of the enzyme and showed comparable protective effects. Diglycerol phosphate, trehalose, and mannosylglyceramide protected the enzyme to a lower extent. Ectoine conferred no protection, and di- myo-inositol phosphate had a strong destabilizing effect. The superior ability of mannosylglycerate to prevent LDH inactivation was accompanied by a higher efficiency in preventing LDH aggregation induced by heat stress. Moreover, mannosylglycerate induced an increase of 4.5 degrees C in the melting temperature of LDH, whereas the same molar concentration of trehalose caused an increase of only 2.2 degrees C. The effectiveness of mannosylglycerate in protecting LDH was also compared to that of other chemically related compounds: mannose, methyl-mannoside, potassium glycerate, glucosylglycerol, glycerol, and glucose. Mannosylglycerate conferred the highest protection, but glucosylglycerol and potassium glycerate were very efficient; glucose exerted a low degree of protection, glycerol and methyl-mannoside had no significant effect, and mannose caused destabilization. Mannosylglycerate was also a good thermoprotectant of glucose oxidase from Aspergillus niger, an enzyme with a net charge opposite to that of LDH under the working conditions. Given the superior performance of mannosylglycerate as a thermoprotectant of enzyme activity in vitro, it is conceivable that it also fulfills a protein thermoprotective function in vivo.     

Biochemical and Immunological Study of Cell Envelope Proteins in Sulfate-Reducing Bacteria

The envelope proteins of 5 strains of the genus Desulfotomaculum and 12 strains of the genus Desulfovibrio were studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. The Desulfovibrio strains exhibited a typical gram-negative cell envelope, whereas the cell envelope of Desulfotomaculum strains appeared to be gram-positive. A close relationship between strains of Desulfotomaculum nigrificans was observed. A comparison between different species of Desulfotomaculum revealed some degree of similarity between Desulfotomaculum nigrificans and Desulfotomaculum ruminis, whereas Desulfotomaculum orientis seemed unique. The strains of Desulfovibrio salexigens were quite different from the strains of the other species of Desulfovibrio. In two of the strains of Desulfovibrio desulfuricans, a species-specific antigen was observed. The strains of Desulfovibrio vulgaris, Desulfovibrio africanus, and Desulfovibrio gigas and one strain of Desulfovibrio desulfuricans exhibited a similar outer membrane protein profile and also showed very similar antigenic reactions.     

An iron tetrahydroporphyrin prosthetic group common to both assimilatory and dissimilatory sulfite reductases

The heme² chromophore of the “assimilatory” E. coli sulfite reductase is an iron-octacarboxylic tetrahydroporphyrin of the isobacteriochlorin type (1). Although the two “dissimilatory” sulfite reductases, desulfoviridin and desulforubidin, from the sulfate reducing bacteria Desulfovibrio gigas and Desulfovibrio desulfuricans (Norway strain), have absorption spectra and reaction products which differ from those of E. coli sulfite reductase, the present studies indicate that they contain prosthetic groups with an organic structure closely similar or identical to that of the E. coli sulfite reductase heme. EPR spectra show high-spin ferriheme in all three enzymes. It is clear, however, that the prosthetic groups must reside in substantially different environments within their respective proteins.     

Phylogenetic studies of two rubredoxins from sulfate reducing bacteria

Summary The sequences of two rubredoxins isolated from the sulfate reducing bacteria:Desulfovibrio vulgaris andDesulfovibrio gigas have been elucidated. They have similar sequences but many more differences occur than would be expected from two bacteria of the same genus. Of the 52 sites, only 37 are occupied by identical residues. The primary structures are compared with those of the anaerobic bacteria rubredoxins ofClostridium pasteurianum, Micrococcus aerogenes, Pseudomonas oleovorans andPeptostreptococcus elsdenii: only 12 identities are found, mostly in the two clusters that contain two iron-bound cysteines each. A phylogenetic tree based on the primary structures is presented and possible relations with plant and bacterial ferredoxins are discussed. A secondary and tertiary structure, stereochemically compatible with the sequence data, is proposed.To whom reprint requests should be addressed     

Methylmercury Resistance in Desulfovibrio desulfuricans Strains in Relation to Methylmercury Degradation

Two strains of Desulfovibrio desulfuricans, one known to synthesize monomethylmercury from ionic mercury, were grown to determine methylmercury toxicity and for comparison with an anaerobic strain of Clostridium pasteurianum, a H₂ producer, and with the broad-spectrum mercury-resistant Pseudomonas putida strain FB-1, capable of degrading 1 μg of methylmercury to methane and elemental mercury in 2 h. The CH₃HgCl resistance of D. desulfuricans strains was 10 times that of P. putida FB-1 and 100 times that of C. pasteurianum. The methylmercury resistance of D. desulfuricans was related to the disappearance of methylmercury from cultures by transformation to dimethylmercury, metacinnabar, methane, and traces of ionic mercury. During a 15-day experiment the kinetics of the two volatile compounds dimethylmercury [(CH₃)₂Hg] and methane were monitored in the liquid by a specific new technique with purge-and-trap gas chromatography in line with Fourier transform infrared spectroscopy and in the headspace by gas chromatography with flame ionization detection. Insoluble metacinnabar (cubic HgS) of biological origin was detected by X-ray diffractometry in the gray precipitate from the insoluble residue of the pellet of a 1-liter culture spiked with 100 mg of CH₃HgCl. This was compared with a 1-liter culture of D. desulfuricans LS spiked with 100 mg of HgCl₂. In a further experiment, it was demonstrated that insoluble, decomposable, white dimethylmercury sulfide [(CH₃Hg)₂S] formed instantly in the reaction of methylmercury with hydrogen sulfide. This organomercurial was extracted with chloroform and identified by gas chromatography in line with mass spectrometry. The D. desulfuricans strains were resistant to high concentrations of methylmercury because they produced insoluble dimethylmercury sulfide, which slowly decomposed under anaerobic conditions to metacinnabar and volatilized to dimethylmercury and methane between pHs 6.2 and 6.5 for high (4.5-g · liter^-1) or low (0.09-g · liter^-1) sulfate contents. Methane was produced from CH₃HgCl at a lower rate than by the broad-spectrum Hg-resistant P. putida strain FB-1.     

Oxygen Consumption by Desulfovibrio Strains with and without Polyglucose

The kinetics of oxygen reduction by Desulfovibrio salexigens Mast1 and the role of polyglucose in this activity were examined and compared with those of strains of D. desulfuricans and D. gigas. Oxidation rates were highest at air saturation (up to 40 nmol of O₂ min⁻¹ mg of protein⁻¹) and declined with decreasing oxygen concentrations. Studies with cell extracts (CE) indicated that NADH oxidase was entirely responsible for the oxygen reduction in strain Mast1. In D. desulfuricans CSN, at least three independent systems appeared to reduce oxygen. Two were active at all oxygen concentrations (NADH oxidase and NADPH oxidase), and one was maximally active at less than 10 μM oxygen. In contrast to D. gigas and D. salexigens strains, the D. desulfuricans strains also contained NADH peroxidase and NADPH peroxidase activities and did not accumulate polyglucose under nonlimiting growth conditions. At air saturation, initial activities of the oxidases and peroxidases of cells harvested at the end of the log phase were on the order of 20 to 140 nmol of O₂ min⁻¹ mg of protein⁻¹. In all strains, these enzymes were relatively stable but were susceptible to inactivation as soon as substrates were added to the assay mixture. Under those conditions, all oxidation activity disappeared after ca. 1 h of incubation. The same finding was observed with whole cells of D. desulfuricans CSN and D. desulfuricans ATCC 27774, but inactivation was less pronounced with cells of D. salexigens Mast1. It appeared that the presence of polyglucose in the whole cells retarded the process of inactivation of NADH oxidase, but this property was lost in crude CE. In spite of the effect of polyglucose on the oxidative potential, oxygen-dependent growth of D. salexigens Mast1 could be demonstrated neither in batch nor in continuous culture.     

Ethanol dissimilation in Desulfovibrio

During growth of ethanol plus sulfate Desulfovibrio gigas and three other Desulfovibrio strains tested contained high NAD-dependent alcohol dehydrogenase activities and dye-linked aldehyde dehydrogenase activities. In lactate-grown cells these activities were lower or absent. In D. gigas an NADH dehydrogenase activity was found which was higher during growth on ethanol than during growth on lactate. The NADH dehydrogenase activity appeared to consist of at least three different soluble enzymes. The aldehyde dehydrogenase activity in D. gigas was highest with benzylviologen as an acceptor and was strongly stimulated by potassium ions. Coenzyme A or phosphate dependency could not be shown, indicating that acetyl-CoA or acetyl phosphate are not intermediates in the conversion of acetaldehyde to acetate.In the absence of sulfate D. gigas was able to convert ethanol to acetate by means of interspecies hydrogen transfer to a methanogen. This conversion, however, did not lead to growth of the Desulfovibrio.Abbreviations DH dehydrogenase - BV²⁺/BV⁺ oxidized/reduced benzylviologen - DCPIP 2,6-dichlorophenolindophenol - MTT 3-(4,5-dimethylthiazol-2-yl)-2,4-diphenyltetrazolium bromide - MV²⁺/MV⁺ oxidized/reduced methylviologen - PMS phenazine methosulfate     

Low-temperature magnetic circular dichroism evidence for the conversion of four-iron-sulphur clusters in a ferredoxin from Clostridium pasteurianum into three-iron-sulphur clusters

Oxidation of the 8Fe ferredoxin from Clostridium pasteurianum with potassium ferricyanide, followed by purification on Sephadex G-25 and DE-23 cellulose columns, gives a protein with an intense EPR signal at g 2.01. The low-temperature magnetic circular dichroism (MCD) spectra of this species are different from those of the oxidized high-potential iron protein from Chromatium but identical with the spectra of ferredoxin II from Desulphovibrio gigas. On reduction of the ferricyanide-treated ferredoxin with sodium dithionite only a weak EPR signal with g factors of 2.05, 1.94 and 1.89 is obtained. The low-temperature MCD spectra are strongly temperature dependent with a form similar to those of dithionite-reduced D. gigas ferredoxin II. The MCD magnetization curves are dominated by a species with ground-state effective g factors of $\text{g}{}_{\mathrm{?}}$ 8.0 and g_⊥ 0.0, which are also similar to those determined recently by low-temperature MCD spectroscopy for D. gigas ferredoxin II. The MCD characteristics are quite different from those of dithionite-reduced ferredoxin from Cl. pasteurianum, untreated with ferricyanide. This establishes the close similarity of the iron-sulphur clusters in ferricyanide-treated Cl. pasteurianum ferredoxin and in D. gigas ferredoxin II. The latter is known to contain a single 3Fe centre, similar to that observed in ferredoxin I from Azotobacter vinelandii by X-ray crystallography. Therefore, it is concluded that the [4Fe-4S] clusters of Cl. pasteurianum ferredoxin are converted to 3Fe clusters on oxidation with ferricyanide.     

Whole-cell antigens of members of the sulfate-reducing genus Desulfovibrio

Antisera have been developed against the wholecell antigens of Desulfovibrio africanus Benghazi and Walvis Bay, D. vulgaris Hildenborough, D. salexigens British Guiana, D. gigas, and D. desulfuricans Essex 6. An enzymelinked immunoadsorption assay (ELISA) was developed to measure the reaction of these antisera with the homologous and heterologous antigens. The ELISA method demonstrated a reaction between pre-immune sera and cells of D. africanus, D. gigas and D. desulfuricans, suggesting the presence of a lectin-like substance on these cell surfaces. Extensive cross-reactions were seen between the antisera and heterologous cells, suggesting the sharing of a number of surface antigens amongst the Desulfovibrio. However, the pattern of these cross-reactions was different from that observed for an ELISA reaction developed for the cytochrome c₃ from various Desulfovibrio.Abbreviation ELISA enzyme-linked immunoadsorption assay     

The relationship between hydrogen metabolism,sulfate reduction and nitrogen fixation in sulfate reducers

Summary Hydrogenase and nitrogenase activities of sulfate-reducing bacteria allow their adaptation to different nutritional habits even under adverse conditions. These exceptional capabilities of adaptation are important factors in the understanding of their predominant role in problems related to anaerobic metal corrosion. Although the D₂–H⁺ exchange reaction indicated thatDesulfovibrio desulfuricans strain Berre-Sol andDesulfovibrio gigas hydrogenases were reversible, the predominant activity in vivo was hydrogen uptake. Hydrogen production was restricted to some particular conditions such as sulfate or nitrogen starvation. Under diazotrophic conditions, a transient hydrogen evolution was followed by uptake when dinitrogen was effectively fixed. In contrast, hydrogen evolution proceeded when acetylene was substituted as the nitrogenase substrate. Hydrogen can thus serve as an electron donor in sulfate reduction and nitrogen metabolism.     

Carbon Flow in Mercury Biomethylation by Desulfovibrio desulfuricans

Radiocarbon incorporation from pyruvate and serine into monomethylmercury by Desulfovibrio desulfuricans was consistent with the proposal that the methyl group originates from C-3 of serine. Immunodiagnostic assays measured 4 to 35 μg of tetrahydrofolate and 58 to 161 ng of cobalamin or a closely related cobalt porphyrin per g of cell protein in D. desulfuricans. The light-reversible inhibition of mercury methylation by propyl iodide in D. desulfuricans indicates methyl transfer by a cobalt porphyrin.     

A new type of metal-binding site in cobalt- and zinc-containing adenylate kinases isolated from sulfate-reducers Desulfovibrio gigas and Desulfovibrio desulfuricans ATCC 27774

Adenylate kinase (AK) mediates the reversible transfer of phosphate groups between the adenylate nucleotides and contributes to the maintenance of their constant cellular level, necessary for energy metabolism and nucleic acid synthesis. The AK were purified from crude extracts of two sulfate-reducing bacteria (SRB), Desulfovibrio (D.) gigas NCIB 9332 and Desulfovibrio desulfuricans ATCC 27774, and biochemically and spectroscopically characterised in the native and fully cobalt- or zinc-substituted forms. These are the first reported adenylate kinases that bind either zinc or cobalt and are related to the subgroup of metal-containing AK found, in most cases, in Gram-positive bacteria. The electronic absorption spectrum is consistent with tetrahedral coordinated cobalt, predominantly via sulfur ligands, and is supported by EPR. The involvement of three cysteines in cobalt or zinc coordination was confirmed by chemical methods. Extended X-ray absorption fine structure (EXAFS) indicate that cobalt or zinc are bound by three cysteine residues and one histidine in the metal-binding site of the “LID” domain. The sequence ¹²⁹Cys-X₅-His-X₁₅-Cys-X₂-Cys of the AK from D. gigas is involved in metal coordination and represents a new type of binding motif that differs from other known zinc-binding sites of AK. Cobalt and zinc play a structural role in stabilizing the LID domain.     

A novel parameterization scheme for energy equations and its use to calculate the structure of protein molecules

A novel scheme for the parameterization of a type of “potential energy” function for protein molecules is introduced. The function is parameterized based on the known conformations of previously determined protein structures and their sequence similarity to a molecule whose conformation is to be calculated. Once parameterized, minima of the potential energy function can be located using a version of simulated annealing which has been previously shown to locate global and near-global minima with the given functional form. As a test problem, the potential was parameterized based on the known structures of the rubredoxins from Desulfovibrio vulgaris, Desulfovibrio desulfuricans, and Clostridium pasteurianum, which vary from 45 to 54 amino acids in length, and the sequence alignments of these molecules with the rubredoxin sequence from Desulfovibrio gigas. Since the Desulfovibrio gigas rubredeoxin conformation has also been determined, it is possible to check the accuracy of the results. Ten simulated-annealing runs from random starting conformations were performed. Seven of the 10 resultant conformations have an all-C_α rms deviation from the crystallographically determined conformation of less than 1.7 Å. For five of the structures, the rms deviation is less than 0.8 Å. Four of the structures have conformations which are virtually identical to each other except for the position of the carboxy-terminal residue. This is also the conformation which is achieved if the determined crystal structure is minimized with the same potential. The all-C_α rms difference between the crystal and minimized crystal structures is 0.6 Å. It is further observed that the “energies” of the structures according to the potential function exhibit a strong correlation with rms deviation from the native structure. The conformations of the individual model structures and the computational aspects of the modeling procedure are discussed. © 1993 Wiley-Liss, Inc.