Thermococcus profundus 2-ketoisovalerate ferredoxin oxidoreductase, a key enzyme in the archaeal energy-producing amino acid metabolic pathway

2-Ketoisovalerate ferredoxin oxidoreductase (VOR) is a key enzyme in hyperthermophiles catalyzing the coenzyme A-dependent oxidative decarboxylation of mainly aliphatic amino acid-derived 2-keto acids. The very oxygen-labile enzyme purified under anaerobic conditions from a hyperthermophilic archaeon, Thermococcus profundus, is a hetero-octamer (alphabetagammadelta)(2) consisting of four different subunits, alpha = 45,000, beta = 31,000, gamma = 22,000 and delta = 13,000, respectively. Electron paramagnetic resonance and resonance Raman spectra of the purified enzyme indicate the presence of approximately three [4Fe-4S] clusters per alphabetagammadelta-protomer, although one of the clusters has a tendency to be converted to a [3Fe-4S] form during purification. The optimal temperature for the enzyme activity is 93 +/- 2 degrees C and the cognate [4Fe-4S] ferredoxin serves as an electron acceptor of the enzyme. The purified enzyme is highly oxygen-labile (t(1/2), approximately 5 min at 25 degrees C), and is partly protected in the presence of magnesium ions, thiamine pyrophosphate and sodium chloride (t(1/2), approximately 25 min at 25 degrees C).     

Characterization of a fourth type of 2-keto acid-oxidizing enzyme from a hyperthermophilic archaeon: 2-ketoglutarate ferredoxin oxidoreductase from Thermococcus litoralis.

Thermococcus litoralis is a strictly anaerobic archaeon (archaebacterium) that grows at temperatures up to 98 degrees C by fermenting peptides. It is known to contain three distinct ferredoxin-dependent, 2-keto acid oxidoreductases, which use pyruvate, aromatic 2-keto acids such as indolepyruvate, or branched-chain 2-keto acids such as 2-ketoisovalerate, as their primary substrates. We show here that T. litoralis also contains a fourth member of this family of enzymes, 2-ketoglutarate ferredoxin oxidoreductase (KGOR). In the presence of coenzyme A, KGOR catalyzes the oxidative decarboxylation of 2-ketoglutarate to succinyl coenzyme A and CO2 and reduces T. litoralis ferredoxin. The enzyme was oxygen sensitive (half-life of approximately 5 min) and was purified under anaerobic conditions. It had an M(r) of approximately 210,000 and appeared to be an octomeric enzyme (alpha2beta2gamma2delta2) with four different subunits with M(r)s of 43,000 (alpha), 29,000 (beta), 23,000 (gamma), and 10,000 (delta). The enzyme contained 0.9 mol of thiamine PPi and at least four [4Fe-4S] clusters per mol of holoenzyme as determined by metal analyses and electron paramagnetic resonance spectroscopy. Significant amounts of other metals (Cu, Zn, Mo, W, and Ni) were not present (<0.1 mol/mol of holoenzyme). Pure KGOR did not utilize other 2-keto acids, such as pyruvate, indolepyruvate, or 2-ketoisovalerate, as substrates, and the apparent Km values for 2-ketoglutarate, coenzyme A, T. litoralis ferredoxin, and thiamine PPi were approximately 250, 40, 8, and 9 microM, respectively. The enzyme was virtually inactive at 25 degrees C and exhibited optimal activity above 90 degrees C (at pH 8.0) and at pH 8.0 (at 80 degrees C). KGOR was quite thermostable, with a half-life at 80 degrees C (under anaerobic conditions) of about 2 days. An enzyme analogous to KGOR has been previously purified from a mesophilic archaeon, but the molecular properties of T. litoralis KGOR more closely resemble those of the other oxidoreductases from hyperthermophiles. In contrast to these enzymes, however, KGOR appears to have a biosynthetic function rather than a role in energy conservation.     

Molecular and phylogenetic characterization of pyruvate and 2-ketoisovalerate ferredoxin oxidoreductases from Pyrococcus furiosus and pyruvate ferredoxin oxidoreductase from Thermotoga maritima.

Previous studies have shown that the hyperthermophilic archaeon Pyrococcus furiosus contains four distinct cytoplasmic 2-ketoacid oxidoreductases (ORs) which differ in their substrate specificities, while the hyperthermophilic bacterium Thermotoga maritima contains only one, pyruvate ferredoxin oxidoreductase (POR). These enzymes catalyze the synthesis of the acyl (or aryl) coenzyme A derivative in a thiamine PPi-dependent oxidative decarboxylation reaction with reduction of ferredoxin. We report here on the molecular analysis of the POR (por) and 2-ketoisovalerate ferredoxin oxidoreductase (vor) genes from P. furiosus and of the POR gene from T. maritima, all of which comprise four different subunits. The operon organization for P. furiosus POR and VOR was porG-vorDAB-porDAB, wherein the gamma subunit is shared by the two enzymes. The operon organization for T. maritima POR was porGDAB. The three enzymes were 46 to 53% identical at the amino acid level. Their delta subunits each contained two ferredoxin-type [4Fe-4S] cluster binding motifs (CXXCXXCXXXCP), while their beta subunits each contained four conserved cysteines in addition to a thiamine PPi-binding domain. Amino-terminal sequence comparisons show that POR, VOR, indolepyruvate OR, and 2-ketoglutarate OR of P. furiosus all belong to a phylogenetically homologous OR family. Moreover, the single-subunit pyruvate ORs from mesophilic and moderately thermophilic bacteria and from an amitochondriate eucaryote each contain four domains which are phylogenetically homologous to the four subunits of the hyperthermophilic ORs (27% sequence identity). Three of these subunits are also homologous to the dimeric POR from a mesophilic archaeon, Halobacterium halobium (21% identity). A model is proposed to account for the observed phenotypes based on genomic rearrangements of four ancestral OR subunits.     

Purifications and characterizations of a ferredoxin and its related 2-oxoacid:ferredoxin oxidoreductase from the hyperthermophilic archaeon, Sulfolobus solfataricus P1

The coenzyme A-acylating 2-oxoacid:ferredoxin oxidoreductase and ferredoxin (an effective electron acceptor) were purified from the hyperthermophilic archaeon, Sulfolobus solfataricus P1 (DSM1616). The purified ferredoxin is a monomeric protein with an apparent molecular mass of approximately 11 kDa by SDS-PAGE and of 11,180+/-50 Da by MALDI-TOF mass spectrometry. Ferredoxin was identified to be a dicluster, [3Fe-4S][4Fe-4S], type ferredoxin by spectrophotometric and EPR studies, and appeared to be zinc-containing based on the shared homology of its N-terminal sequence with those of known zinc-containing ferredoxins. On the other hand, the purified 2-oxoacid: ferredoxin oxidoreductase was found to be a heterodimeric enzyme consisting of 69 kDa alpha and 34 kDa beta subunits by SDS-PAGE and MALDI-TOF mass spectrometry. The purified enzyme showed a specific activity of 52.6 units/mg for the reduction of cytochrome c with 2-oxoglutarate as substrate at 55 degrees C, pH 7.0. Maximum activity was observed at 70 degrees C and the optimum pH for enzymatic activity was 7.0 -8.0. The enzyme displays broad substrate specificity toward 2-oxoacids, such as pyruvate, 2-oxobutyrate, and 2-oxoglutarate. Among the 2-oxoacids tested (pyruvate, 2-oxobutyrate, and 2-oxoglutarate), 2-oxoglutarate was found to be the best substrate with Km and kcat values of 163 microM and 452 min(-1), respectively. These results provide useful information for structural studies on these two proteins and for studies on the mechanism of electron transfer between the two.     

Purification and characterization of the tungsten enzyme aldehyde:ferredoxin oxidoreductase from the hyperthermophilic denitrifier Pyrobaculum aerophilum

A tungsten-containing aldehyde:ferredoxin oxidoreductase (AOR) has been purified to homogeneity from Pyrobaculum aerophilum. The N-terminal sequence of the isolated enzyme matches a single open reading frame in the genome. Metal analysis and electron paramagnetic resonance (EPR) spectroscopy indicate that the P. aerophilum AOR contains one tungsten center and one [4Fe-4S]^2+/1+ cluster per 68-kDa monomer. Native AOR is a homodimer. EPR spectroscopy of the purified enzyme that has been reduced with the substrate crotonaldehyde revealed a W(V) species with g_zyx values of 1.952, 1.918, 1.872. The substrate-reduced AOR also contains a [4Fe-4S]¹⁺ cluster with S=3/2 and zero field splitting parameters D=7.5 cm^–1 and E/D=0.22. Molybdenum was absent from the enzyme preparation. The P. aerophilum AOR lacks the amino acid sequence motif indicative for binding of mononuclear iron that is typically found in other AORs. Furthermore, the P. aerophilum AOR utilizes a 7Fe ferredoxin as the putative physiological redox partner, instead of a 4Fe ferredoxin as in Pyrococcus furiosus. This 7Fe ferredoxin has been purified from P. aerophilum, and the amino acid sequence has been identified using mass spectrometry. Direct electrochemistry of the ferredoxin showed two one-electron transitions, at –306 and –445 mV. In the presence of 55 M ferredoxin the AOR activity is 17% of the activity obtained with 1 mM benzyl viologen as an electron acceptor.     

Biochemical characterization of pantoate kinase, a novel enzyme necessary for coenzyme a biosynthesis in the archaea

Although bacteria and eukaryotes share a pathway for coenzyme A (CoA) biosynthesis, we previously clarified that most archaea utilize a distinct pathway for the conversion of pantoate to 4'-phosphopantothenate. Whereas bacteria/eukaryotes use pantothenate synthetase and pantothenate kinase (PanK), the hyperthermophilic archaeon Thermococcus kodakarensis utilizes two novel enzymes: pantoate kinase (PoK) and phosphopantothenate synthetase (PPS). Here, we report a detailed biochemical examination of PoK from T. kodakarensis. Kinetic analyses revealed that the PoK reaction displayed Michaelis-Menten kinetics toward ATP, whereas substrate inhibition was observed with pantoate. PoK activity was not affected by the addition of CoA/acetyl-CoA. Interestingly, PoK displayed broad nucleotide specificity and utilized ATP, GTP, UTP, and CTP with comparable k(cat)/K(m) values. Sequence alignment of 27 PoK homologs revealed seven conserved residues with reactive side chains, and variant proteins were constructed for each residue. Activity was not detected when mutations were introduced to Ser104, Glu134, and Asp143, suggesting that these residues play vital roles in PoK catalysis. Kinetic analysis of the other variant proteins, with mutations S28A, H131A, R155A, and T186A, indicated that all four residues are involved in pantoate recognition and that Arg155 and Thr186 play important roles in PoK catalysis. Gel filtration analyses of the variant proteins indicated that Thr186 is also involved in dimer assembly. A sequence comparison between PoK and other members of the GHMP kinase family suggests that Ser104 and Glu134 are involved in binding with phosphate and Mg(2+), respectively, while Asp143 is the base responsible for proton abstraction from the pantoate hydroxy group.     

Purification, characterization, and metabolic function of tungsten-containing aldehyde ferredoxin oxidoreductase from the hyperthermophilic and proteolytic archaeon Thermococcus strain ES-1.

Thermococcus strain ES-1 is a strictly anaerobic, hyperthermophilic archaeon that grows at temperatures up to 91 degrees C by the fermentation of peptides. It is obligately dependent upon elemental sulfur (S(o)) for growth, which it reduces to H2S. Cell extracts contain high aldehyde oxidation activity with viologen dyes as electron acceptors. The enzyme responsible, which we term aldehyde ferredoxin oxidoreductase (AOR), has been purified to electrophoretic homogeneity. AOR is a homodimeric protein with a subunit M(r) of approximately 67,000. It contains molybdopterin and one W, four to five Fe, one Mg, and two P atoms per subunit. Electron paramagnetic resonance analyses of the reduced enzyme indicated the presence of a single [4Fe-4S]+ cluster with an S = 3/2 ground state. While AOR oxidized a wide range of aliphatic and aromatic aldehydes, those with the highest apparent kcat/Km values (> 10 microM-1S-1) were acetaldehyde, isovalerylaldehyde, and phenylacetaldehyde (Km values of < 100 microM). The apparent Km value for Thermococcus strain ES-1 ferredoxin was 10 microM (with crotonaldehyde as the substrate). Thermococcus strain ES-1 AOR also catalyzed the reduction of acetate (apparent Km of 1.8 mM) below pH 6.0 (with reduced methyl viologen as the electron donor) but at much less than 1% of the rate of the oxidative reaction (with benzyl viologen as the electron acceptor at pH 6.0 to 10.0). The properties of Thermococcus strain ES-1 AOR are very similar to those of AOR previously purified from the saccharolytic hyperthermophile Pyrococcus furiosus, in which AOR was proposed to oxidize glyceraldehyde as part of a novel glycolytic pathway (S. Mukund and M. W. W. Adams, J. Biol. Chem. 266:14208-14216, 1991). However, Thermococcus strain ES-1 is not known to metabolize carbohydrates, and glyceraldehyde was a very poor substrate (kcat/Km of < 0.2 microM-1S-1) for its AOR. The most efficient substrates for Thermococcus strain ES-1 AOR were the aldehyde derivatives of transaminated amino acids. This suggests that the enzyme functions to oxidize aldehydes generated during amino acid catabolism, although the possibility that AOR generates aldehydes from organic acids produced by fermentation cannot be ruled out.     

The ferredoxin-binding site of ferredoxin: Nitrite oxidoreductase. Differential chemical modification of the free enzyme and its complex with ferredoxin.

Spinach (Spinacea oleracea) leaf ferredoxin (Fd)-dependent nitrite reductase was treated with either the arginine-modifying reagent phenyl-glyoxal or the lysine-modifying reagent pyridoxal-5'-phosphate under conditions where only the Fd-binding affinity of the enzyme was affected and where complex formation between Fd and the enzyme prevented the inhibition by either reagent. Modification with [14C]phenylglyoxal allowed the identification of two nitrite reductase arginines, R375 and R556, that are protected by Fd against labeling. Modification of nitrite reductase with pyridoxal-5'-phosphate, followed by reduction with NaBH4, allowed the identification of a lysine, K436, that is protected by Fd against labeling. Positive charges are present at these positions in all of the Fd-dependent nitrite reductase for which sequences are available, suggesting that these amino acids are directly involved in electrostatic binding of Fd to the enzyme.     

Characterization of the 2-phospho-L-lactate transferase enzyme involved in coenzyme F(420) biosynthesis in Methanococcus jannaschii

The protein product of the Methanococcus jannaschii MJ1256 gene has been expressed in Escherichia coli, purified to homogeneity, and shown to be involved in coenzyme F(420) biosynthesis. The protein catalyzes the transfer of the 2-phospholactate moiety from lactyl (2) diphospho-(5')guanosine (LPPG) to 7,8-didemethyl-8-hydroxy-5-deazariboflavin (Fo) with the formation of the L-lactyl phosphodiester of 7,8-didemethyl-8-hydroxy-5-deazariboflavin (F(420)-0) and GMP. On the basis of the reaction catalyzed, the enzyme is named LPPG:Fo 2-phospho-L-lactate transferase. Since the reaction is the fourth step in the biosynthesis of coenzyme F(420), the enzyme has been designated as CofD, the product of the cofD gene. The transferase requires Mg(2+) for activity, and the catalysis does not appear to proceed via a covalent intermediate. To a lesser extent CofD also catalyzes a number of additional reactions that include the formation of Fo-P, when the enzyme is incubated with Fo and GDP, GTP, pyrophosphate, or tripolyphosphate, and the hydrolysis of F(420)-0 to Fo. All of these side reactions can be rationalized as occurring by a common mechanism. CofD has no recognized sequence similarity to any previously characterized enzyme.     

Inactivation of clostridial ferredoxin and pyruvate-ferredoxin oxidoreductase by sodium nitrite.

Clostridial ferredoxin and pyruvate-ferredoxin oxidoreductase activity was investigated after in vitro or in vivo treatment with sodium nitrite. In vitro treatment of commercially available Clostridium pasteurianum ferredoxin with sodium nitrite inhibited ferredoxin activity. Inhibition of ferredoxin activity increased with increasing levels of sodium nitrite. Ferredoxin was isolated from normal C. pasteurianum and Clostridium botulinum cultures and from cultures incubated with 1,000 micrograms of sodium nitrite per ml for 45 min. The activity of in vivo nitrite-treated ferredoxin was decreased compared with that of control ferredoxin. Pyruvate-ferredoxin oxidoreductase isolated from C. botulinum cultures incubated with 1,000 micrograms of sodium nitrite per ml showed less activity than did control oxidoreductase. It is concluded that the antibotulinal activity of nitrite is due at least in part to inactivation of ferredoxin and pyruvate-ferredoxin oxidoreductase.     

The novel tungsten-iron-sulfur protein of the hyperthermophilic archaebacterium, Pyrococcus furiosus, is an aldehyde ferredoxin oxidoreductase. Evidence for its participation in a unique glycolytic pathway

The anaerobic archaebacterium, Pyrococcus furiosus, grows optimally at 100 degrees C by a fermentative-type metabolism in which H2, CO2, and organic acids are end products. The growth of this organism is stimulated by tungsten, and, from it, a novel, red-colored, tungsten-iron-sulfur protein, abbreviated RTP, has been purified (Mukund, S., and Adams, M. W. W. (1990) J. Biol. Chem. 265, 11508-11516). RTP (Mr approximately 85,000) contained approximately 1W, 7Fe, and 5 acid-labile sulfide atoms/molecule and exhibited unique EPR properties. The physiological function of the protein, however, was unknown. We show here that RTP is an inactive form of an aldehyde ferredoxin oxidoreductase (AOR). The active enzyme was obtained by rapid purification under anaerobic conditions using buffers containing dithiothreitol and glycerol. AOR catalyzed the oxidation of a range of aliphatic aldehydes with an optimum temperature for activity above 90 degrees C, but it did not oxidize glucose or glyceraldehyde 3-phosphate, nor reduce NAD(P), and its activity was independent of CoA. The active (AOR) and inactive (RTP) forms of the enzyme were indistinguishable in their contents of metals and acid-labile sulfide and in their EPR properties. The latter are though to originate from two nonidentical and spin-coupled iron-sulfur clusters, whereas the tungsten in this enzyme, which was not detectable by EPR, appears to be present as a novel pterin cofactor. Inhibition and activation studies indicated that AOR contains a catalytically essential W-SH group that is not present in RTP, the inactive form. AOR is a new type of aldehyde-oxidizing enzyme and is the first aldehyde oxidoreductase to be purified from an archaebacterium or a nonactogenic anaerobic bacterium. Its physiological role in P. furiosus is proposed as the oxidation of glyceraldehyde to glycerate in a unique, partially nonphosphorylated, glycolytic pathway that generates acetyl-CoA from glucose without the participation of nicotinamide nucleotides.     

Three-dimensional structure of a new enzyme, O-phosphoserine sulfhydrylase, involved in l-cysteine biosynthesis by a hyperthermophilic archaeon, Aeropyrum pernix K1, at 2.0A resolution

O-Phosphoserine sulfhydrylase is a new enzyme found in a hyperthermophilic archaeon, Aeropyrum pernix K1. This enzyme catalyzes a novel cysteine synthetic reaction from O-phospho-l-serine and sulfide. The crystal structure of the enzyme was determined at 2.0A resolution using the method of multi-wavelength anomalous dispersion. A monomer consists of three domains, including an N-terminal domain with a new alpha/beta fold. The topology folds of the middle and C-terminal domains were similar to those of the O-acetylserine sulfhydrylase-A from Salmonella typhimurium and the cystathionine beta-synthase from human. The cofactor, pyridoxal 5'-phosphate, is bound in a cleft between the middle and C-terminal domains through a covalent linkage to Lys127. Based on the structure determined, O-phospho-l-serine could be rationally modeled into the active site of the enzyme. An enzyme-substrate complex model and a mutation experiment revealed that Arg297, unique to hyperthermophilic archaea, is one of the most crucial residues for O-phosphoserine sulfhydrylation activity. There are more hydrophobic areas and less electric charges at the dimer interface, compared to the S.typhimurium O-acetylserine sulfhydrylase.     

Sulfide dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus: a new multifunctional enzyme involved in the reduction of elemental sulfur.

Pyrococcus furiosus is an anaerobic archaeon that grows optimally at 100 degrees C by the fermentation of carbohydrates yielding acetate, CO2, and H2 as the primary products. If elemental sulfur (S0) or polysulfide is added to the growth medium, H2S is also produced. The cytoplasmic hydrogenase of P. furiosus, which is responsible for H2 production with ferredoxin as the electron donor, has been shown to also catalyze the reduction of polysulfide to H2S (K. Ma, R. N. Schicho, R. M. Kelly, and M. W. W. Adams, Proc. Natl. Acad. Sci. USA 90:5341-5344, 1993). From the cytoplasm of this organism, we have now purified an enzyme, sulfide dehydrogenase (SuDH), which catalyzes the reduction of polysulfide to H2S with NADPH as the electron donor. SuDH is a heterodimer with subunits of 52,000 and 29,000 Da. SuDH contains flavin and approximately 11 iron and 6 acid-labile sulfide atoms per mol, but no other metals were detected. Analysis of the enzyme by electron paramagnetic resonance spectroscopy indicated the presence of four iron-sulfur centers, one of which was specifically reduced by NADPH. SuDH has a half-life at 95 degrees C of about 12 h and shows a 50% increase in activity after 12 h at 82 degrees C. The pure enzyme has a specific activity of 7 mumol of H2S produced.min-1.mg of protein-1 at 80 degrees C with polysulfide (1.2 mM) and NADPH (0.4 mM) as substrates. The apparent Km values were 1.25 mM and 11 microM, respectively. NADH was not utilized as an electron donor for polysulfide reduction. P. furiosus rubredoxin (K(m) = 1.6 microM) also functioned as an electron acceptor for SuDH, and SuDH catalyzed the reduction of NADP with reduced P. furiosus ferredoxin (K(m) = 0.7 microM) as an electron donor. The multiple activities of SuDH and its proposed role in the metabolism of S(o) and polysulfide are discussed.     

Engineering a three-cysteine, one-histidine ligand environment into a new hyperthermophilic archaeal Rieske-type [2Fe-2S] ferredoxin from Sulfolobus solfataricus

We heterologously overproduced a hyperthermostable archaeal low potential (E(m) = -62 mV) Rieske-type ferredoxin (ARF) from Sulfolobus solfataricus strain P-1 and its variants in Escherichia coli to examine the influence of ligand substitutions on the properties of the [2Fe-2S] cluster. While two cysteine ligand residues (Cys(42) and Cys(61)) are essential for the cluster assembly and/or stability, the contributions of the two histidine ligands to the cluster assembly in the archaeal Rieske-type ferredoxin appear to be inequivalent as indicated by much higher stability of the His(64) --> Cys variant (H64C) than the His(44) --> Cys variant (H44C). The x-ray absorption and resonance Raman spectra of the H64C variant firmly established the formation of a novel, oxidized [2Fe-2S] cluster with one histidine and three cysteine ligands in the archaeal Rieske-type protein moiety. Comparative resonance Raman features of the wild-type, natural abundance and uniformly (15)N-labeled ARF and its H64C variant showed significant mixing of the Fe-S and Fe-N stretching characters for an oxidized biological [2Fe-2S] cluster with partial histidine ligation.     

Characterization of Arabidopsis thaliana pinoresinol reductase, a new type of enzyme involved in lignan biosynthesis

A lignan, lariciresinol, was isolated from Arabidopsis thaliana, the most widely used model plant in plant bioscience sectors, for the first time. In the A. thaliana genome database, there are two genes (At1g32100 and At4g13660) that are annotated as pinoresinol/lariciresinol reductase (PLR). The recombinant AtPLRs showed strict substrate preference toward pinoresinol but only weak or no activity toward lariciresinol, which is in sharp contrast to conventional PLRs of other plants that can reduce both pinoresinol and lariciresinol efficiently to lariciresinol and secoisolariciresinol, respectively. Therefore, we renamed AtPLRs as A. thaliana pinoresinol reductases (AtPrRs). The recombinant AtPrR2 encoded by At4g13660 reduced only (-)-pinoresinol to (-)-lariciresinol and not (+)-pinoresinol in the presence of NADPH. This enantiomeric selectivity accords with that of other PLRs of other plants so far reported, which can reduce one of the enantiomers selectively, whatever the preferential enantiomer. In sharp contrast, AtPrR1 encoded by At1g32100 reduced both (+)- and (-)-pinoresinols to (+)- and (-)-lariciresinols efficiently with comparative k(cat)/K(m) values. Analysis of lignans and spatiotemporal expression of AtPrR1 and AtPrR2 in their functionally deficient A. thaliana mutants and wild type indicated that both genes are involved in lariciresinol biosynthesis. In addition, the analysis of the enantiomeric compositions of lariciresinol isolated from the mutants and wild type showed that PrRs together with a dirigent protein(s) are involved in the enantiomeric control in lignan biosynthesis. Furthermore, it was demonstrated conclusively for the first time that differential expression of PrR isoforms that have distinct selectivities of substrate enantiomers can determine enantiomeric compositions of the product, lariciresinol.     

Tetrahydromethanopterin,a coenzyme involved in autotrophic acetyl coenzyme A synthesis from 2 CO2 in Methanobacterium

Methanobacterium thermoautotrophicum, a methane forming archaebacterium, grows autotrophically by synthesizing activated acetic acid from 2 CO₂. It is demonstrated in vitro that the methyl group of acetate is derived from methenyl tetrahydromethanopterin, which is known to be a one-carbon carrying coenzyme in CO₂ reduction to methane. The direct acetate precursors are suggested to be methyl tetrahydromethanopterin (“activated methanol”) and “activated carbon monoxide”.     

Comparative analysis of pyruvate kinases from the hyperthermophilic archaea Archaeoglobus fulgidus,Aeropyrum pernix,and Pyrobaculum aerophilum and the hyperthermophilic bacterium Thermotoga maritima: unusual regulatory properties in hyperthermophilic archaea

Pyruvate kinases (PK, EC 2.7.1.40) from three hyperthermophilic archaea (Archaeoglobus fulgidus strain 7324, Aeropyrum pernix, and Pyrobaculum aerophilum) and from the hyperthermophilic bacterium Thermotoga maritima were compared with respect to their thermophilic, kinetic, and regulatory properties. PKs from the archaea are 200-kDa homotetramers composed of 50-kDa subunits. The enzymes required divalent cations, Mg2+ and Mn2+ being most effective, but were independent of K+. Temperature optima for activity were 85 degrees C (A. fulgidus) and above 98 degrees C (A. pernix and P. aerophilum). The PKs were highly thermostable up to 110 degrees C (A. pernix) and showed melting temperatures for thermal unfolding at 93 degrees C (A. fulgidus) or above 98 degrees C (A. pernix and P. aerophilum). All archaeal PKs exhibited sigmoidal saturation kinetics with phosphoenolpyruvate (PEP) and ADP indicating positive homotropic cooperative response with both substrates. Classic heterotropic allosteric regulators of PKs from eukarya and bacteria, e.g. fructose 1,6-bisphosphate or AMP, did not affect PK activity of hyperthermophilic archaea, suggesting the absence of heterotropic allosteric regulation. PK from the bacterium T. maritima is also a homotetramer of 50-kDa subunits. The enzyme was independent of K+ ions, had a temperature optimum of 80 degrees C, was highly thermostable up to 90 degrees C, and had a melting temperature above 98 degrees C. The enzyme showed cooperative response to PEP and ADP. In contrast to its archaeal counterparts, the T. maritima enzyme exhibited the classic allosteric response to the activator AMP and to the inhibitor ATP. Sequences of hyperthermophilic PKs showed significant similarity to characterized PKs from bacteria and eukarya. Phylogenetic analysis of PK sequences of all three domains indicates a distinct archaeal cluster that includes the PK from the hyperthermophilic bacterium T. maritima.     

Purification and characterization of two reversible and ADP-dependent acetyl coenzyme A synthetases from the hyperthermophilic archaeon Pyrococcus furiosus.

Pyrococcus furiosus is a strictly anaerobic archaeon (archaebacterium) that grows at temperatures up to 105 degrees C by fermenting carbohydrates and peptides. Cell extracts have been previously shown to contain an unusual acetyl coenzyme A (acetyl-CoA) synthetase (ACS) which catalyzes the formation of acetate and ATP from acetyl-CoA by using ADP and phosphate rather than AMP and PPi. We show here that P. furiosus contains two distinct isoenzymes of ACS, and both have been purified. One, termed ACS I, uses acetyl-CoA and isobutyryl-CoA but not indoleacetyl-CoA or phenylacetyl-CoA as substrates, while the other, ACS II, utilizes all four CoA derivatives. Succinyl-CoA did not serve as a substrate for either enzyme. ACS I and ACS II have similar molecular masses (approximately 140 kDa), and both appear to be heterotetramers (alpha2beta2) of two different subunits of 45 (alpha) and 23 (beta) kDa. They lack metal ions such as Fe2+, Cu2+, Zn2+, and Mg2+ and are stable to oxygen. At 25 degrees C, both enzymes were virtually inactive and exhibited optimal activities above 90 degrees C (at pH 8.0) and at pH 9.0 (at 80 degrees C). The times required to lose 50% of their activity at 80 degrees C were about 18 h for ACS I and 8 h for ACS II. With both enzymes in the acid formation reactions, ADP and phosphate could be replaced by GDP and phosphate but not by CDP and phosphate or by AMP and PPi. The apparent Km values for ADP, GDP, and phosphate were approximately 150, 132, and 396 microM, respectively, for ACS I (using acetyl-CoA) and 61, 236, and 580 microM, respectively, for ACS II (using indoleacetyl-CoA). With ADP and phosphate as substrates, the apparent Km values for acetyl-CoA and isobutyryl-CoA were 25 and 29 microM, respectively, for ACS I and 26 and 12 microM, respectively, for ACS II. With ACS II, the apparent Km value for phenylacetyl-CoA was 4 microM. Both enzymes also catalyzed the reverse reaction, the ATP-dependent formation of the CoA derivatives of acetate (I and II), isobutyrate (I and II), phenylacetate (II only), and indoleacetate (II only). The N-terminal amino acid sequences of the two subunits of ACS I were similar to those of ACS II and to that of a hypothetical 67-kDa protein from Escherichia coli but showed no similarity to mesophilic ACS-type enzymes. To our knowledge, ACS I and II are the first ATP-utilizing enzymes to be purified from a hyperthermophile, and ACS II is the first enzyme of the ACS type to utilize aromatic CoA derivatives.