Electron paramagnetic resonance and other properties of hydrogenases isolated from Desulfovibrio vulgaris (strain Hildenborough) and Megasphaera elsdenii

The hydrogenases of Desulfovibrio vulgaris and Megasphaera elsdenii are compared with respect to some of their physical properties. In addition to Fe the only metal ions that are present in significant amounts are Ni and Cu. From cluster extrusion experiments it follows that the D. vulgaris enzyme contains three 4 Fe-4S clusters, while M. elsdenii hydrogenase only releases part of its Fe-S clusters. The resting D. vulgaris enzyme shows only a small 3 Fe-xS type of EPR signal (maximum 5% electron equivalent). This amount can be increased to approximately 25% by treatment with ferricyanide, with a concomitant large decrease in activity. The M. elsdenii enzyme shows in its oxidized state a normal Hipip (high-potential iron-sulphur protein) type of EPR spectrum. After a reduction/oxidation cycle the D. vulgaris enzyme also shows a weak Hipip type of EPR spectrum. In the reduced state both enzymes show complex spectra. By integration of those spectra it is shown that 1.5 electron equivalents are present. The complex spectra do not arise from nuclear hyperfine interactions but are partially due to electron spin interactions. It is proposed that the spectrum of reduced D. vulgaris hydrogenase consists of a sum of three different ferredoxin-like spectra.     

Purification and properties of hydrogenase from Megasphaera elsdenii

A hydrogenase has been purified to homogeneity from the soluble fraction of the rumen bacterium Megasphaera elsdenii, the overall purification is 200 times with a yield of 14%. The pure enzyme consists of a single polypeptide chain with Mr approximately 50 000 which contains 12 atoms of non-haem iron and 12 atoms of acid-labile sulphide. The enzyme is rapidly inactivated by O2 and it is therefore purified under nitrogen and in the presence of sodium dithionite. The optical spectrum of the enzyme, after removal of the dithionite with air, shows a peak at 275 nm (epsilon 275 nm = 143 mM-1 cm-1) and a shoulder between 350 nm and 400 nm (epsilon 400 nm = 46 mM-1 cm-1). The enzyme catalyses hydrogen production from sodium dithionite at a low rate. The rate is greatly enhanced by addition of the electron donors flavodoxin, ferredoxin and methyl viologen. The kinetic data with these three electron donors suggest co-operativity, but no indication of self-association of the enzyme was obtained. Sodium chloride enhances the rate of hydrogen production with methyl viologen semiquinone and changes the kinetic behaviour of the enzyme with this electron donor, but causes inhibition of the reactions mediated by ferredoxin and flavodoxin. Two kinetic models were developed which are consistent with the kinetic data of the three electron donors tested. The apparent co-operativity for the hydrogen production can be fitted with the mathematical form of those models. The identical kinetic behaviour of the hydrogenase with the one-electron donors flavodoxin and methyl viologen semiquinone monomer and the two-electron donor ferredoxin indicates that the hydrogenase accepts two electrons in two separate, independent steps and further indicates that the two (4Fe-4S) clusters of the donor ferredoxin are independent. The interpretation of the kinetic data with methyl viologen semiquinone is complicated by the fact that the semiquinone dimerises, and that the formation of the dimer is enhanced by salt. Taking into account the association of this donor, the activity of the enzyme with methyl viologen semiquinone can be described by the sum of the activities of the enzyme with methyl viologen monomer and methyl viologen dimer. The enzyme catalyses the oxidation of hydrogen gas with methyl and benzyl viologen as electron acceptors to their semiquinone forms; both electron acceptors show Michaelis-Menten kinetics. The hydrogen oxidation activity with both electron acceptors is stimulated by addition of sodium chloride. The kinetic data of the oxidation of hydrogen with the two-electron acceptors used are consistent with the porposed models, if it is assumed that the pathway followed is compulsory. At this moment no choice can be made between the models proposed.     

Redox properties of electron-transferring flavoprotein from Megasphaera elsdenii

Electron-transferring flavoprotein (ETF) from the anaerobic bacterium Megasphaera elsdenii catalyzes electron transfer from NADH or D-lactate dehydrogenase to butyryl-CoA dehydrogenase. As a basis for understanding the interactions of ETF with its substrates, we report here on the redox properties of ETF alone. ETF exhibited reversible, two-electron transfer during electrochemical reduction in the presence of mediator dyes. The midpoint redox potentials of the FAD cofactor were -0.185 V at pH 5.5, -0.259 V at pH 7.1 and -0.269 +/- 0.013 V at pH 8.4, all versus the standard hydrogen electrode In the presence of the indicator dye 1-deazariboflavin, the Nernst slopes were 0.029 V and 0.026 V at pH 5.5 and pH 7.1, respectively, compared with an expected value of 0.028 V at 10 degrees C. At pH 8.4, in the presence of 2-hydroxy-1,4-naphthoquinone or phenosafranine, the Nernst slope varied from 0.021 V to 0.041 V. In the experiments at pH 8.4, equilibration was very slow in the reductive direction and a difference of as much as 30 mV was observed between reductive and oxidative midpoints. ETF exhibited no thermodynamic stabilization of the radical form of the FAD cofactor during electrochemical reduction at pH 5.5, 7.1 or 8.4. However, up to 93% of kinetically stable, anionic radical was produced by dithionite titration at pH 8.5. Molar absorptivities of ETF radical were 17,000 M-1 X cm-1 at 365 nm and 5100 M-1 X cm-1 at 450 nm. The four ETF preparations used here contained less than 7% 6-OH-FAD. However, two of the preparations contained significant amounts (up to 30%) of flavin which stabilized radical and reduced at potentials 0.2 V more positive than those required for reduction of the major form of ETF. This is referred to as the B form of ETF. The proportion of ETF-FAD in the B form was increased by incubation with free FAD or by a cycle of reduction and reoxidation. These treatments caused marked changes in the absorption spectrum of oxidized ETF and decreases of 20-25% in ETF units/A450.     

Characterization of a reserve glucan from Megasphaera elsdenii.

A reserve glucan of Megasphaera elsdenii was studied by methylation analysis before and after treatment with isoamylase. The results of this study indicate that the glucan is of the amylopectin-glycogen type.     

Genome sequence of the ruminal bacterium Megasphaera elsdenii

Megasphaera elsdenii is a Gram-negative ruminal bacterium. It is being investigated as a probiotic supplement for ruminants as it may provide benefits for energy balance and animal productivity. Furthermore, it is of biotechnological interest due to its capability of producing various volatile fatty acids. Here we report the complete genome sequence of M. elsdenii DSM 20460, the type strain for the species.     

Characterization and heterologous expression of plasmalogen synthase MeHAD from Megasphaera elsdenii

Plasmalogens (Pls) are vinyl-ether bond-containing glycerophospholipids or glycosyl diradyl glycerols, and are of great importance in the physiological functions and stability of cell membrane. Here, we identified and characterized that the plasmalogen synthase MeHAD from anaerobic Megasphaera elsdenii was responsible for vinyl-ether bond formation. Different from the 2-hydroxyacyl-CoA dehydratase (HAD) family plasmalogen synthase PlsA-PlsR which are encoded by two genes in Clostridium perfringens, the HAD homolog (MeHAD) encoded by a single gene MELS_0169 was found in M. elsdenii. By heterologous expression of the MeHAD gene into a nonplasmalogen-producing Escherichia coli strain, the expressed MeHAD was found to be located in the cell membrane region. Plasmalogens were detected in the recombinant strain using GC–MS and LC-MS, demonstrating that MeHAD was the key enzyme for plasmalogen synthesis. Moreover, the synthesized plasmalogens could enhance the oxidative stress-resistance and osmotic pressure-resistance of the recombinant strain, probably due to the ROS scavenging and decreased membrane permeability by the plasmalogens, respectively. The four-cysteine (Cys125, Cys164, Cys445 and Cys484) site-mutant of MeHAD, which were predicted binding to the [4Fe-4S] cluster, was unable to synthesize plasmalogens, indicating that the cysteines are important for the catalytic activity of MeHAD. Our results revealed the single gene encoded plasmalogen synthase in M. elsdenii and established a recombinant E. coli strain with plasmalogen production potential.     

Purification and Properties of a Hydrogenase from Desulfovibrio vulgaris

The soluble hydrogenase of Desulfovibrio vulgaris was purified and some of its properties are described. The molecular weight was determined for the enzyme by sedimentation equilibrium (45,400) and amino acid analysis (44,800). The hydrogenase appears to be a loosely coiled molecule or to have a high axial ratio, which is reflected in an unusually low sedimentation coefficient (2.58S) and a low diffusion coefficient (D 5.85). The molecular weight of the hydrogenase (41,000), as calculated by the Svedberg equation, was in general agreement with the sedimentation equilibrium molecular weight. Amino acid analysis revealed the presence of six halfcystine residues per mole of enzyme and a total of 417 amino acid residues. The specificity of the hydrogenase and its capacity to reduce certain low potential dyes and cytochrome c(3) was studied. Metal analysis of the hydrogenase indicated the presence of 1 mole of ferrous iron per mole of enzyme.     

Butyryl-CoA dehydrogenase from Megasphaera elsdenii. Specificity of the catalytic reaction.

The absorption coefficient of butyryl-CoA dehydrogenase from Megasphaera elsdenii at 450 nm is determined as 14.4 mM-1 X cm-1 in the CoA-free form and 14.2 mM-1 X cm-1 in the CoA-liganded form (both yellow). The latter value is considerably higher than the earlier published estimate. Phenazine ethosulphate offers great advantages over phenazine methosulphate as a coupling dye in the catalytic assay despite giving lower Vmax. values (506 min-1 as compared with 1250 min-1 under the conditions used). The phenazine ethosulphate assay is used to establish a pH optimum of 8.05 for oxidation of 100 microM-butyryl-CoA. The rates of oxidation of a range of straight-chain, branched-chain and alicyclic acyl thioesters are used to provide the following information. Only straight-chain acyl groups containing 4-6 carbon atoms are easily accommodated by the postulated hydrophobic pocket of the enzyme. C-3-substituted acyl-CoA thioesters are not oxidized at a significant rate, suggesting that the C-3 pro-S-hydrogen atom of straight-chain substrates is partially exposed to the solvent. Acyl-CoA thioesters with substitutions at C-2 are oxidized, though at a lower rate than their straight-chain counterparts. This implies that the C-2 pro-S-hydrogen atom of straight-chain substrates is partially exposed to the solvent. Saturated alicyclic carboxylic acyl-CoA thioesters with 4-7 carbon atoms in the ring are oxidized, with maximal activity for the cyclohexane derivative. This implies that optimal oxidation requires a true trans orientation of the two departing hydrogen atoms. The strain imposed by bound unsaturated alicyclic acyl thioesters strikingly perturbs the flavin visible-absorption spectrum, with the exception of the cyclohex-2-ene derivative, which forms a complex with similar spectral properties to those of the crotonyl-CoA complex. In the thiol moiety of thioester substrates the amide bond of N-acetylcysteamine is essential for both binding and catalysis. The adenosine structure contributes substantially to strong binding, but is less important in determining the catalytic rate.     

Isolation of Acidaminococcus fermentans and Megasphaera elsdenii from Normal Human Feces

Fecal bacterial cultures from 40 normal humans yielded Megasphaera elsdenii from four individuals and Acidaminococcus fermentans from 10 individuals, with two individuals having both organisms.     

Characterization of the gene encoding the [Fe]-hydrogenase from Megasphaera elsdenii

The gene encoding the [Fe]-hydrogenase from the anaerobic bacterium Megasphaera elsdenii has been cloned and sequenced. The gene is monocistronic, in keeping with the protein being a monomer. The translated protein sequence (484 residues, M(r)=53 kDa) comprises a small 2[4Fe-4S] ferredoxin-like domain and a large domain containing the catalytic site. Comparisons with other [Fe]-hydrogenase sequences, including two of which the crystal structures are known, show that the M. elsdenii protein is among the smallest of these enzymes and provide useful indications regarding the basic structural core common to all [Fe]-hydrogenases. It is, nevertheless, to be noted that the genome of Thermotoga maritima encodes a putative [Fe]-hydrogenase that would consist of only 301 residues.     

Purification and properties of the flavoenzyme D-lactate dehydrogenase from Megasphaera elsdenii.

A pyridine nucleotide independent D-lactate dehydrogenase has been purified to apparent homogeneity from the anaerobic bacterium Megasphaera elsdenii. The enzyme has a molecular weight of 105 000 by sedimentation equilibrium analysis with a subunit molecular weight of 55 000 by sodium dodecyl sulfate gel electrophoresis and is thus probably a dimer of identical subunits. It contains approximately 1 mol of FAD and 1 g-atom of Zn2+ per mol of protein subunit, and the flavin exhibits a fluorescence 1.7 times that of free FAD. An earlier purification [Brockman, H. L., & Wood, W. A. (1975 J. Bacteriol. 124, 1454--1461] results in substantial loss of the enzyme's zinc, which is required for catalytic activity. The new purification yields greater than 5 times the amount of enzyme previously isolated. The enzyme is specific for D-lactate, and no inhibition is observed with L-lactate. Surprisingly, the enzyme has a significant oxidase activity, which depends on the ionic strength. Vmax values of 190 and 530 min-1 were obtained at a gamma/2 of 0.224 and 0.442, respectively. Except for this atypically high oxygen reactivity, D-lactate dehydrogenase resembles other flavoenzyme dehydrogenases in that the flavin does not react with sulfite, the tryptophan content is low, and a neutral blue semiquinone is formed upon photochemical reduction. The enzyme flavin is reduced either by dithionite, by oxalate plus catalytic 5-deazaflavin in the presence of light, or by D-lactate. Two electrons per flavin were consumed in a dithionite titration, implyine with varying ratios of D-lactate and pyruvate, an Em7 of -0.219 +/- 0.007 V at 20 degrees C was calculated for the flavin. The enzyme requires dithiothreitol for stability. Rapid inactivation results when the enzyme is incubated with a substoichiometric level of Cu2+. This inactivation can be reversed by dithiothreitol. It is proposed that the enzyme possesses a pair of cysteine residues capable of facile disulfide formation.     

Purification and Properties of Sulfite Reductase from Desulfovibrio vulgaris,Miyazaki F

The sulfite reductase of Desulfovibrio vulgaris, strain Miyazaki F (MF), was purified by ammonium sulfate precipitation and chromatography on DEAE-cellulose, Ultrogel AcA34, and hydroxylapatite. The molecular weight was estimated to be 180,000 by gel filtration. It had a subunit structure of α₂β₂; the molecular weight of the α subunit was 50,000 and that of β, 39,000. The absorption spectrum with characteristic peaks at 629 and 409 nm and the amino acid composition resembled those of the sulfite reductase from D. vulgaris, Miyazaki K. The MF enzyme reduced sulfite to trithionate, thiosulfate, and sulfide by hydrogen when coupled with a hydrogenase-methyl viologen system, like other sulfite reductases from Desulfovibrio.     

Crystal structure of Saccharomyces cerevisiae 3'-phosphoadenosine-5'-phosphosulfate reductase complexed with adenosine 3',5'-bisphosphate

Most assimilatory bacteria, fungi, and plants species reduce sulfate (in the activated form of APS or PAPS) to produce reduced sulfur. In yeast, PAPS reductase reduces PAPS to sulfite and PAP. Despite the difference in substrate specificity and catalytic cofactor, PAPS reductase is homologous to APS reductase in both sequence and structure, and they are suggested to share the same catalytic mechanism. Metazoans do not possess the sulfate reduction pathway, which makes APS/PAPS reductases potential drug targets for human pathogens. Here, we present the 2.05 A resolution crystal structure of the yeast PAPS reductase binary complex with product PAP bound. The N-terminal region mediates dimeric interactions resulting in a unique homodimer assembly not seen in previous APS/PAPS reductase structures. The "pyrophosphate-binding" sequence (47)TTAFGLTG(54) defines the substrate 3'-phosphate binding pocket. In yeast, Gly54 replaces a conserved aspartate found in APS reductases vacating space and charge to accommodate the 3'-phosphate of PAPS, thus regulating substrate specificity. Also, for the first time, the complete C-terminal catalytic motif (244)ECGIH(248) is revealed in the active site. The catalytic residue Cys245 is ideally positioned for an in-line attack on the beta-sulfate of PAPS. In addition, the side chain of His248 is only 4.2 A from the Sgamma of Cys245 and may serve as a catalytic base to deprotonate the active site cysteine. A hydrophobic sequence (252)RFAQFL(257) at the end of the C-terminus may provide anchoring interactions preventing the tail from swinging away from the active site as seen in other APS/PAPS reductases.     

Metabolism and some characteristics of ruminal strains of Megasphaera elsdenii

Megasphaera elsdenii belongs to the group comprising the ruminal and intestinal lactate- and sugar-fermenting species. In the present study the fermentation characteristics, metabolism of glucose and lactate, and susceptibility to antimicrobial agents of four ruminal strains were investigated. Particular attention was given to the mixed-substrate fermentation pattern and resultant fermentation acid profile. Lactate was utilized more rapidly than glucose in media with both carbon sources. Interaction of the two substrates changed the composition of fermentation end products toward more valerate and less propionate in cultures with glucose and lactate. Contrary to the indications in Bergey's Manual of Systematic Bacteriology, butyrate, not caproate, was the main end product of glucose metabolism. The strains examined were rather insensitive to many antimicrobial compounds, especially to ionophores and other antimicrobial feed additives.     

Occurrence of adenosine 2',5'-bisphosphate in rat liver

Adenosine 2',5'-bisphosphate (pAp) is present in liver from 2-day-fasted rats, at a concentration of around 1 microM. pAp was obtained through perchloric acid extraction of the liver followed by two successive DEAE-cellulose chromatographies and an ion-pair high-pressure liquid chromatography. Both pAp extracted from liver and that obtained from a commercial source showed the same pattern of hydrolysis by alkaline phosphatase, i.e., more 5'-AMP than 2'-AMP was obtained as an intermediate of the reaction.     

Photochemical labeling of bovine pancreatic ribonuclease A with 8-azidoadenosine 3',5'-bisphosphate

A simple method has been developed for the preparation of 5'-32P-labeled 8-azidoadenosine 3',5'-bisphosphate (p8N3Ap) for use in photoaffinity labeling studies. Irradiation of a complex between p8N3Ap and bovine pancreatic ribonuclease A (RNase A) with light of 300-350 nm led to the covalent attachment of the nucleotide to the enzyme. RNase A could also be labeled in the dark with prephotolyzed p8N3Ap. In either case, the nucleotide reacted with the same tryptic peptide, encompassing amino acids 67-85 of the protein. The site of labeling was determined to be either Thr-78 or Thr-82, both of which are close to or at the pyrimidine binding site of the enzyme. This result is consistent with recent nuclear magnetic resonance and X-ray studies which indicate that 8-substituted adenine nucleotides interact with the pyrimidine binding site of RNase A.     

Creation of salt-insensitive 3'(2'),5'-bisphosphate nucleotidase by modeling and mutagenesis approach

3′(2′),5′-Bisphosphate nucleotidase, (EC 3.1.3.7) (BPntase) is a ubiquitous enzyme. Recently, these enzymes have drawn considerable attention as in vivo targets of salt toxicity as well as therapeutic targets of lithium that is used for the treatment of manic-depressive disorders. They belong to the Mg²⁺-dependent Li⁺-sensitive phosphomonoesterase super-family and are highly sensitive to lithium and sodium ions. However, the molecular mechanism of inhibition of this group of enzymes by monovalent cations has not been completely understood. Previously we have identified a BPntase (Dhal2p) from a highly halotolerant yeast Debaryomyces hansenii. Molecular characterization revealed a number of unique features in Dhal2p, indicating this is an extraordinary member of the family. In this study, we have carried out the structure-function analysis of Dhal2p through the combination of molecular modeling and in vitro mutagenesis approach. We have not only provided the explanation for the role played by the functionally important elements that are conserved among the members of this family but also identified important, novel structural elements in this enzyme. Our study for the first time unraveled the role of a flap as well as a loop region in the functioning of this enzyme. Most importantly, mutations in the loop region resulted in the creation of a BPntase that was insensitive to salt.     

Hydrodynamic, structural and magnetic properties of Megasphaera elsdenii Fe hydrogenase reinvestigated

Megasphaera elsdenii hydrogenase has been purified to homogeneity using an FPLC procedure as the final step. The protein gives a single band in SDS/PAGE with an apparent molecular mass of 57-59 kDa. There is no second hydrogenase activity in the soluble fraction of M. elsdenii. The hydrodynamics of the enzyme have been compared to those of the two-subunit Fe hydrogenase from Desulfovibrio vulgaris (Hildenborough) in the analytical ultracentrifuge using the absorption of the intrinsic iron-sulfur clusters as the monitor. Sedimentation-velocity experiments indicate the M. elsdenii enzyme (s20,w = 4.95 S) to be essentially globular, while the D. vulgaris enzyme (s20,w = 4.1 S) has a less symmetric shape. From the sedimentation equilibrium measurements under a variety of conditions an average molecular mass is calculated of 58 kDa (M. elsdenii) and 54 kDa (D. vulgaris), respectively. Pure, maximally active M. elsdenii hydrogenase has A405/A280 = 0.36 and has a specific H2-production activity of 400 mumol H2.min-1.(mg protein)-1 at 30 degrees C and pH 8.0. The enzyme contains some 13-18 iron and acid-labile sulfur ions/58-kDa monomer. Eight of these Fe-S are present as two electron-transferring ferredoxin-like cubanes with Em approximately greater than -0.3 V, as indicated by pH-dependent EPR spectroscopy on the H2-reduced enzyme. In the (re)oxidized state the remainder iron gives rise to a single S = 1/2 rhombic EPR signal. Hydrogen-production activity, content of remainder iron and rhombic EPR signal intensity are mutually correlated. Purified hydrogenase appears to exist as a mixture of fully active holoenzyme and inactive protein still carrying the two cubanes but deficient in active-site iron.     

Properties of purified hydrogenase from the particulate fraction of Desulfovibrio vulgaris, Miyazaki.

The properties of purified hydrogenase [EC 1.12.2.1] solubilized from particulate fraction of sonicated Desulfovibrio vulgaris cells are described. The enzyme was a brownish iron-sulfur protein of molecular weight 89,000, composed of two different subunits (mol. wt.: 28,000 and 59,000), and it contained 7-9 iron atoms and 7-8 labile sulfide ions. Molybdenum was not detected in the preparation. The absorption spectrum of the enzyme was characteristic of iron-sulfur proteins. The millimolar absorbance coefficients of the enzyme were about 164 at 280nm, and 47 at 400nm. The absorption spectrum of the enzyme in the visible region changed upon incubating the enzyme under H2 in the presence of cytochrome c3, but not in its absence. This spectral change was due to the reduction of the enzyme. The absorbance ratio at 400nm of the reduced and the oxidized forms of the enzyme was 0.66. The activity of the enzyme was hardly affected by metal-complexing agents such as cyanide, azide, 1,10-phenanthroline, etc., except for CO, which was a strong inhibitor of the enzyme. The activity was inhibited by SH-reagents such as p-chloromercuribenzenesulfonate. The enzyme was significantly resistant to urea, but susceptible to sodium dodecyl sulfate. These properties were very similar to those of clostridial hydrogenase [EC 1.12.7.1], in spite of differences in the acceptor specificity and subunit structure.