Acetyl-CoA decarbonylase/synthase complex from Archaeoglobus fulgidus

The acetyl-CoA decarbonylase/synthase (ACDS) multienzyme complex catalyzes the reversible cleavage and synthesis of acetyl-CoA in methanogens. This report of the enzyme complex in Archaeoglobus fulgidus demonstrates the existence of a functional ACDS complex in an organism that is not a methanogen. The A. fulgidus enzyme complex contained five subunits of 89, 72, 50, 49.5, and 18.5 kDa, and it catalyzed the overall synthesis of acetyl-CoA according to the following reaction: w CO₂ + 2 Fd_red(Fe²⁺) + 2 H⁺ + CH₃– H₄SPt + CoA ⇌ acetyl-CoA + H₄SPt + 2 Fd_ox(Fe³⁺) + H₂O where Fd is ferredoxin, and CH₃–H₄SPt and H₄SPt denote N ⁵-methyl-tetrahydrosarcinapterin and tetrahydrosarcinapterin, respectively. Received: 27 October 1997 / Accepted: 29 January 1998     

Menaquinone-specific prenyl reductase from the hyperthermophilic archaeon Archaeoglobus fulgidus

Four genes that encode the homologues of plant geranylgeranyl reductase were isolated from a hyperthermophilic archaeon Archaeoglobus fulgidus, which produces menaquinone with a fully saturated heptaprenyl side chain, menaquinone-7(14H). The recombinant expression of one of the homologues in Escherichia coli led to a distinct change in the quinone profile of the host cells, although the homologue is the most distantly related to the geranylgeranyl reductase. The new compounds found in the profile had successively longer elution times than those of ordinary quinones from E. coli, i.e., menaquinone-8 and ubiquinone-8, in high-performance liquid chromatography on a reversed-phase column. Structural analyses of the new compounds by electron impact-mass spectrometry indicated that their molecular masses progressively increase relative to the ordinary quinones at a rate of 2 U but that they still contain quinone head structures, strongly suggesting that the compounds are quinones with partially saturated prenyl side chains. In vitro assays with dithionite as the reducing agent showed that the prenyl reductase is highly specific for menaquinone-7, rather than ubiquinone-8 and prenyl diphosphates. This novel enzyme noncovalently binds flavin adenine dinucleotide, similar to geranylgeranyl reductase, but was not able to utilize NAD(P)H as the electron donor, unlike the plant homologue.     

Dual coenzyme specificity of Archaeoglobus fulgidus HMG-CoA reductase

Comparison of the inferred amino acid sequence of orf AF1736 of Archaeoglobus fulgidus to that of Pseudomonas mevalonii HMG-CoA reductase suggested that AF1736 might encode a Class II HMG-CoA reductase. Following polymerase chain reaction-based cloning of AF1736 from A. fulgidus genomic DNA and expression in Escherichia coli, the encoded enzyme was purified to apparent homogeneity and its enzymic properties were determined. Activity was optimal at 85 degrees C, deltaHa was 54 kJ/mol, and the statin drug mevinolin inhibited competitively with HMG-CoA (Ki 180 microM). Protonated forms of His390 and Lys277, the apparent cognates of the active site histidine and lysine of the P. mevalonii enzyme, appear essential for activity. The mechanism proposed for catalysis of P. mevalonii HMG-CoA reductase thus appears valid for A. fulgidus HMG-CoA reductase. Unlike any other HMG-CoA reductase, the A. fulgidus enzyme exhibits dual coenzyme specificity. pH-activity profiles for all four reactions revealed that optimal activity using NADP(H) occurred at a pH from 1 to 3 units more acidic than that observed using NAD(H). Kinetic parameters were therefore determined for all substrates for all four catalyzed reactions using either NAD(H) or NADP(H). NADPH and NADH compete for occupancy of a common site. k(cat)[NAD(H)]/k(cat)[NADP(H)] varied from unity to under 70 for the four reactions, indicative of slight preference for NAD(H). The results indicate the importance of the protonated status of active site residues His390 and Lys277, shown by altered K(M) and k(cat) values, and indicate that NAD(H) and NADP(H) have comparable affinity for the same site.     

Characterization of a catalase-peroxidase from the hyperthermophilic archaeon Archaeoglobus fulgidus

A putative perA gene from Archaeoglobus fulgidus was cloned and expressed in Escherichia coli BL21(DE3), and the recombinant catalase-peroxidase was purified to homogeneity. The enzyme is a homodimer with a subunit molecular mass of 85 kDa. UV-visible spectroscopic analysis indicated the presence of protoheme IX as a prosthetic group (ferric heme), in a stoichiometry of 0.25 heme per subunit. Electron paramagnetic resonance analysis confirmed the presence of ferric heme and identified the proximal axial ligand as a histidine. The enzyme showed both catalase and peroxidase activity with pH optima of 6.0 and 4.5, respectively. Optimal temperatures of 70 degrees C and 80 degrees C were found for the catalase and peroxidase activity, respectively. The catalase activity strongly exceeded the peroxidase activity, with Vmax values of 9600 and 36 U mg(-1), respectively. Km values for H2O2 of 8.6 and 0.85 mM were found for catalase and peroxidase, respectively. Common heme inhibitors such as cyanide, azide, and hydroxylamine inhibited peroxidase activity. However, unlike all other catalase-peroxidases, the enzyme was also inhibited by 3-amino-1,2,4-triazole. Although the enzyme exhibited a high thermostability, rapid inactivation occurred in the presence of H2O2, with half-life values of less than 1 min. This is the first catalase-peroxidase characterized from a hyperthermophilic microorganism.     

Archaeoglobus fulgidus Isolated from Hot North Sea Oil Field Waters

A hyperthermophilic sulfate reducer, strain 7324, was isolated from hot (75 degrees C) oil field waters from an oil production platform in the Norwegian sector of the North Sea. It was enriched on a complex medium and isolated on lactate with sulfate. The cells were nonmotile, irregular coccoid to disc shaped, and 0.3 to 1.0 mum wide. The temperature for growth was between 60 and 85 degrees C with an optimum of 76 degrees C. Lactate, pyruvate, and valerate plus H(2) were utilized as carbon and energy sources with sulfate as electron acceptor. Lactate was completely oxidized to CO(2). The cells contained an active carbon monoxide dehydrogenase but no 2-oxoglutarate dehydrogenase activity, indicating that lactate was oxidized to CO(2) via the acetyl coenzyme A/carbon monoxide dehydrogenase pathway. The cells produced small amounts of methane simultaneously with sulfate reduction. F(420) was detected in the cells which showed a blue-green fluorescence at 420 nm. On the basis of morphological, physiological, and serological features, the isolate was classified as an Archaeoglobus sp. Strain 7324 showed 100% DNA-DNA homology with A. fulgidus Z, indicating that it belongs to the species A. fulgidus. Archaeoglobus sp. has been selectively enriched and immunomagnetically captured from oil field waters from three different platforms in the North Sea. Our results show that strain 7324 may grow in oil reservoirs at 70 to 85 degrees C and contribute to hydrogen sulfide formation in this environment.     

Crystal structure of a glycyl radical enzyme from Archaeoglobus fulgidus

We have solved the crystal structure of a PFL2 from Archaeglobus fulgidus at 2.9 A resolution. Of the three previously solved enzyme structures of glycyl radical enzymes, pyruvate formate lyase (PFL), anaerobic ribonucleotide reductase and glycerol dehydratase (GD), the last one is clearly most similar to PFL2. We observed electron density in the active site of PFL2, which we modelled as glycerol. The orientation of the glycerol is different from that in GD, and changes in the active site indicate that the actual substrate of PFL2 is bigger than a glycerol molecule, but sequence and structural homology suggest that PFL2 may be a dehydratase. Crystal packing, solution X-ray scattering and ultracentrifugation experiments show that PFL2 is tetrameric, unlike other glycyl radical enzymes. A.fulgidus is a hyperthermophile and PFL2 appears to be stabilized by several factors including an increased number of ion pairs, differences in buried charges, a truncated N terminus, anchoring of loops and N terminus via salt-bridges, changes in the oligomeric interface and perhaps also the higher oligomerization state of the protein.     

A thermostable shikimate 5-dehydrogenase from the archaeon Archaeoglobus fulgidus

Shikimate 5-dehydrogenase (SKDH; EC 1.1.1.25) catalyzes the reversible reduction of 3-dehydroshikimate to shikimate and is a key enzyme in the aromatic amino acid biosynthesis pathway. The shikimate 5-dehydrogenase gene, aroE, from Archaeoglobus fulgidus was cloned and overexpressed in Escherichia coli. The recombinant enzyme purified as a homodimer and yielded a maximum specific activity of 732 U/mg at 87 degrees C (with NADP+ as coenzyme). Apparent Km values for shikimate, NADP+, and NAD+ were estimated at 0.17+/-0.03 mM, 0.19+/-0.01 mM, and 11.4+/-0.4 mM, respectively. The half-life of the A. fulgidus SKDH is 2 h at the assay temperature (87 degrees C) and 17 days at 60 degrees C. Addition of 1 M NaCl or KCl stabilized the enzyme's half-life to approximately 70 h at 87 degrees C and approximately 50 days at 60 degrees C. This work presents the first kinetic analysis of an archaeal SKDH.     

Structures of the iron-sulfur flavoproteins from Methanosarcina thermophila and Archaeoglobus fulgidus

Iron-sulfur flavoproteins (ISF) constitute a widespread family of redox-active proteins in anaerobic prokaryotes. Based on sequence homologies, their overall structure is expected to be similar to that of flavodoxins, but in addition to a flavin mononucleotide cofactor they also contain a cubane-type [4Fe:4S] cluster. In order to gain further insight into the function and properties of ISF, the three-dimensional structures of two ISF homologs, one from the thermophilic methanogen Methanosarcina thermophila and one from the hyperthermophilic sulfate-reducing archaeon Archaeoglobus fulgidus, were determined. The structures indicate that ISF assembles to form a tetramer and that electron transfer between the two types of redox cofactors requires oligomerization to juxtapose the flavin mononucleotide and [4Fe:4S] cluster bound to different subunits. This is only possible between different monomers upon oligomerization. Fundamental differences in the surface properties of the two ISF homologs underscore the diversity encountered within this protein family.     

Cellular localization of D-lactate dehydrogenase and NADH oxidase from Archaeoglobus fulgidus

Members of the genus Archaeoglobus are hyperthermophilic sulfate reducers with an optimal growth temperature of 83 degrees C. Archaeoglobus fulgidus can utilize simple compounds including D-lactate, L-lactate and pyruvate as the sole substrate for carbon and electrons for dissimilatory sulfate reduction. Previously we showed that this organism makes a D-lactate dehydrogenase (Dld) that requires FAD and Zn2+ for activity. To determine the cellular location and topology of Dld and to identify proteins that interact with Dld, an antibody directed against Dld was prepared. Immunocytochemical studies using gold particle-coated secondary antibodies show that more than 85% of Dld is associated with the membrane. A truncated form of Dld was detected in immunoblots of whole cells treated with protease, showing that Dld is an integral membrane protein and that a significant portion of Dld, including part of the FAD-binding pocket, is outside the membrane facing the S-layer. The gene encoding Dld is part of an operon that includes noxA2, which encodes one of several NADH oxidases in A. fulgidus. Previous studies have shown that NoxA2 remains bound to Dld during purification. Thin sections of A. fulgidus probed simultaneously with antibodies against Dld and NoxA2 show that both proteins co-localized to the same sites in the membrane. Although these data show a tight interaction between NoxA2 and Dld, the role of NoxA2 in electron transport reactions is unknown. Rather, NoxA2 may protect proteins involved in electron transfer by reducing O2 to H2O2 or H2O.     

Role of metal-binding domains of the copper pump from Archaeoglobus fulgidus

CopA from the extreme thermophile Archaeoglobus fulgidus is a P-type ATPase that transports Cu(+) and Ag(+) and has individual metal-binding domains (MBDs) at both N- and C-termini. We expressed and purified full-length CopA as well as constructs with MBDs deleted either individually or collectively. Cu(+) and Ag(+)-dependent ATPase assays showed that full-length CopA had submicromolar affinity for both ions, but was inhibited by concentrations above 1muM. Deletion of both MBDs had no effect on affinity but resulted in loss of this inhibition. Individual deletions implicated the N-terminal MBD in causing the inhibition at concentrations >1muM. Rates of phosphoenzyme decay indicated that neither the dephosphorylation step, nor the E1P-E2P equilibrium accounted for this inhibition, suggesting the involvement of a different catalytic step. Alternative hypotheses are discussed by which the N-terminal MBD could influence the catalytic activity of CopA.     

Functional multiplicity of phosphoglucose isomerase from Lactobacillus casei

Phosphoglucose isomerase (D-glucose-6-phosphate ketolisomerase, EC 5.3.1.9), purified from Lactobacillus casei, showed multiplicity with respect to electrophoretic mobility, molecular weight, kinetic properties and responses to erythrose 4-phosphate. Among the three forms isolated, one having a dimeric conformation, was specific for glucose 6-phosphate. Erythrose 4-phosphate inhibited this preparation in a sigmoid fashion, while this compound activated the enzyme for isomerization of ribose 5-phosphate. In tetrameric conformation of the similar subunits, the enzyme was more specific for ribose 5-phosphate and the inhibition exerted by erythrose 4-phosphate was hyperbolic. The possible implications of these observations have been discussed.     

Inositol-1-phosphate synthase from Archaeoglobus fulgidus is a class II aldolase

A gene putatively identified as the Archaeoglobus fulgidus inositol-1-phosphate synthase (IPS) gene was overexpressed to high level (about 30-40% of total soluble cellular proteins) in Escherichia coli. The recombinant protein was purified to homogeneity by heat treatment followed by two column chromatographic steps. The native enzyme was a tetramer of 168 +/- 4 kDa (subunit molecular mass of 44 kDa). At 90 degrees C the K(m) values for glucose-6-phosphate and NAD(+) were estimated as 0.12 +/- 0.04 mM and 5.1 +/- 0.9 microM, respectively. Use of (D)-[5-(13)C]glucose-6-phosphate as a substrate confirmed that the stereochemistry of the product of the IPS reaction was L-myo-inositol-1-phosphate. This archaeal enzyme, with the highest activity at its optimum growth temperature among all IPS reported (k(cat) = 9.6 +/- 0.4 s(-1) with an estimated activation energy of 69 kJ/mol), was extremely heat stable. However, the most unique feature of A. fulgidus IPS was that it absolutely required divalent metal ions for activity. Zn(2+) and Mn(2+) were the best activators with K(D) approximately 1 microM, while NH(4)(+) (a critical activator for all the other characterized IPS enzymes) had no effect on the enzyme. These properties suggested that this archaeal IPS was a class II aldolase. In support of this, stoichiometric reduction of NAD(+) to NADH could be followed spectrophotometrically when EDTA was present along with glucose-6-phosphate.     

Hypothetical protein AF2241 from Archaeoglobus fulgidus adopts a cyclophilin-like fold

AF2241 is a hypothetical protein from Archaeoglobus fulgidus and it belongs to the PFam domain of unknown function 369 (DUF369). NMR structural determination reveals that AF2241 adopts a cyclophilin-like fold, with a β-barrel core composed of eight β-strands, one α-helix, and one 3₁₀ helix located at each end of the barrel. The protein displays a high structural similarity to TM1367, another member of DUF369 whose structure has been determined recently by X-ray crystallography. Structural similarity search shows that AF2241 also has a high similarity to human cyclophilin A, however, sequence alignment and electrostatic potential analysis reveal that the residues in the PPIase catalytic site of human cyclophilin A are not conserved in AF2241 or TM1367. Instead, a putative active site of AF2241 maps to a negatively charged pocket composed of 9 conserved residues. Our results suggest that although AF2241 adopts the same fold as the human cyclophilin A, it may have distinct biological function. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Characterization and sequence comparison of temperature-regulated chaperonins from the hyperthermophilic archaeon Archaeoglobus fulgidus

We have cloned and sequenced the genes encoding two chaperonin subunits (Cpn-α and Cpn-β), from Archaeoglobus fulgidus, a sulfate-reducing hyperthermophilic archaeon. The genes encode proteins of 545 amino acids with calculated M_r of 58 977 and 59 683. Both proteins have been identified in cytoplasmic fractions of A. fulgidus by Western analysis using antibodies raised against one of the subunits expressed in Escherichia coli, and by N-terminal amino acid sequencing of chaperonin complexes purified by immunoprecipitation. The chaperonin genes appear to be under heat shock regulation, as both proteins accumulate following temperature shift-up of growing A. fulgidus cells, implying a role of the chaperonin in thermoadaptation. Canonical Box A and Box B archaeal promoter sequences, as well as additional conserved putative signal sequences, are located upstream of the start codons. A phylogenetic analysis using all the available archaeal chaperonin sequences, suggests that the α and β subunits are the results of late gene duplications that took place well after the establishment of the main archaeal evolutionary lines.     

Oligomerization behavior of the archaeal Sm2-type protein from Archaeoglobus fulgidus

As part of a functional analysis of archaeal Sm-related proteins, we have studied the oligomerization behavior of the Sm-2 type protein from the euryarchaeon Archaeoglobus fulgidus using gel filtration chromatography and noncovalent mass spectrometry. Our experiments show that the oligomeric state of the protein depends on the pH and presence of RNA. The protein forms a hexamer at acidic pH in the absence of RNA. The addition of RNA (oligo U10) induces the formation of a heptamer over the whole pH range studied. The stability of both the hexamer and the RNA-bound heptamer increases at lower pH.     

Superoxide reduction mechanism of Archaeoglobus fulgidus one-iron superoxide reductase

Superoxide reductases (SORs), iron-centered enzymes responsible for reducing superoxide (O2(-)) to hydrogen peroxide, are found in many anaerobic and microaerophilic prokaryotes. The rapid reaction with an exogenous electron donor renders the reductase activity catalytic. Here, we demonstrate using pulse radiolysis that the initial reaction between O2(-) and Archaeoglobus fulgidus neelaredoxin, a one-iron SOR, leads to a short-lived transient that immediately disappears to yield a solvent-bound ferric species in acid-base equilibrium. Through comparison of wild-type neelaredoxin with mutants lacking the ferric ion coordinating glutamate, we demonstrate that the remaining step is related to the final coordination of this ligand to the oxidized metal center and kinetically characterize it for the first time, by pulse radiolysis and stopped-flow kinetics. The way exogenous phosphate perturbs the kinetics of superoxide reduction by neelaredoxin and mutant proteins was also investigated.