Search

Iron is an essential nutrient for almost all known organisms, but in aerobic, neutral pH environments, it is present primarily as precipitated oxyhydroxide minerals. In contrast, in anaerobic environments, iron can exist in its ferrous form (Fe2+) and remain soluble. In sulfide-rich, anaerobic environments, Fe2+ and sulfide react to form iron sulfide cluster complexes of the form FexSx (FeSaq), which further condense to form the mineral mackinawite, which itself is partly soluble. However, the ability of microorganisms to utilize iron sulfide as an iron source is not known. Here, we show that the anaerobic, hyperthermophilic archaeon Pyrococcus furiosus can directly assimilate the iron in dissolved iron sulfide cluster complexes (FeSaq) without further dissolution to Fe2+. Growth is only inhibited in the presence of a Fe2+-specific chelator. The FeSaq that is utilized can be formed either by reaction of chelated Fe2+ with sulfide or dissolved from mackinawite. Pyrococcus furiosus can utilize FeSaq larger than 3.5 kDa, or Fe40S40, and may actively aid in the dissolution of mackinawite to the assimilated FeSaq. A model for iron sulfide assimilation from an insoluble mineral is proposed.

This study reports how the length of capping ligands on a nanocrystal surface affects its interfacial electron transfer (ET) with surrounding molecular electron acceptors, and consequently, impact the H2 production of a biotic-abiotic hybrid artificial photosynthetic system. Specifically, we study how the H2 production efficiency of a hybrid system, combining CdS nanorods (NRs), [NiFe] hydrogenase, and redox mediators (propyl-bridged 2,2'-bipyridinium, PDQ2+), depends on the alkyl chain length of mercaptocarboxylate ligands on the NR surface. We observe a minor decrease of the quantum yield for H2 production from 54 ± 6 to 43 ± 2% when varying the number of methylene units in the ligands from 2 to 7. In contrast, an abrupt decrease of the yield was observed from 43 ± 2 to 4 ± 1% when further increasing n from 7 to 11. ET studies reveal that the intrinsic ET rates from the NRs to the electron acceptor PDQ2+ are all within 108-109 s-1 regardless of the length of the capping ligands. However, the number of adsorbed PDQ2+ molecules on NR surfaces decreases dramatically when n ≥ 10, with the saturating number changing from 45 ± 5 to 0.3 ± 0.1 for n = 2 and 11, respectively. These results are not consistent with the commonly perceived exponential dependence of ET rates on the ligand length. Instead, they can be explained by the change of the accessibility of NR surfaces to electron acceptors from a disordered "liquid" phase at n ∼7. These results highlight that the order of capping ligands is an important design parameter for further constructing nanocrystal/molecular assemblies in broad nanocrystal-based applications.

The key difference in the modified Embden–Meyerhof glycolytic pathway in hyperthermophilic Archaea, such as Pyrococcus furiosus, occurs at the conversion from glyceraldehyde-3-phosphate (GAP) to 3-phosphoglycerate (3-PG) where the typical intermediate 1,3-bisphosphoglycerate (1,3-BPG) is not present. The absence of the ATP-yielding step catalyzed by phosphoglycerate kinase (PGK) alters energy yield, redox energetics, and kinetics of carbohydrate metabolism. Either of the two enzymes, ferredoxin-dependent glyceraldehyde-3-phosphate ferredoxin oxidoreductase (GAPOR) or NADP+-dependent non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN), responsible for this “bypass” reaction, could be deleted individually without impacting viability, albeit with differences in native fermentation product profiles. Furthermore, P. furiosus was viable in the gluconeogenic direction (growth on pyruvate or peptides plus elemental sulfur) in a ΔgapnΔgapor strain. Ethanol was utilized as a proxy for potential heterologous products (e.g., isopropanol, butanol, fatty acids) that require reducing equivalents (e.g., NAD(P)H, reduced ferredoxin) generated from glycolysis. Insertion of a single gene encoding the thermostable NADPH-dependent primary alcohol dehydrogenase (adhA) (Tte_0696) from Caldanaerobacter subterraneus, resulted in a strain producing ethanol via the previously established aldehyde oxidoreductase (AOR) pathway. This strain demonstrated a high ratio of ethanol over acetate (> 8:1) at 80 °C and enabled ethanol production up to 85 °C, the highest temperature for bio-ethanol production reported to date.

Background Biological conversion of lignocellulosic biomass is significantly hindered by feedstock recalcitrance, which is typically assessed through an enzymatic digestion assay, often preceded by a thermal and/or chemical pretreatment. Here, we assay 17 lines of unpretreated transgenic black cottonwood (Populus trichocarpa) utilizing a lignocellulose-degrading, metabolically engineered bacterium, Caldicellulosiruptor bescii. The poplar lines were assessed by incubation with an engineered C. bescii strain that solubilized and converted the hexose and pentose carbohydrates to ethanol and acetate. The resulting fermentation titer and biomass solubilization were then utilized as a measure of biomass recalcitrance and compared to data previously reported on the transgenic poplar samples. Results Of the 17 transgenic poplar lines examined with C. bescii, a wide variation in solubilization and fermentation titer was observed. While the wild type poplar control demonstrated relatively high recalcitrance with a total solubilization of only 20% and a fermentation titer of 7.3 mM, the transgenic lines resulted in solubilization ranging from 15 to 79% and fermentation titers from 6.8 to 29.6 mM. Additionally, a strong inverse correlation (R2 = 0.8) between conversion efficiency and lignin content was observed with lower lignin samples more easily converted and solubilized by C. bescii. Conclusions Feedstock recalcitrance can be significantly reduced with transgenic plants, but finding the correct modification may require a large sample set to identify the most advantageous genetic modifications for the feedstock. Utilizing C. bescii as a screening assay for recalcitrance, poplar lines with down-regulation of coumarate 3-hydroxylase 3 (C3H3) resulted in the highest degrees of solubilization and conversion by C. bescii. One such line, with a growth phenotype similar to the wild-type, generated more than three times the fermentation products of the wild-type poplar control, suggesting that excellent digestibility can be achieved without compromising fitness of the tree.

Tungsten (W) is a metal that is generally thought to be seldom used in biology. We show here that a W-containing oxidoreductase (WOR) family is diverse and widespread in the microbial world. Surprisingly, WORs, along with the tungstate-specific transporter Tup, are abundant in the human gut microbiome, which contains 24 phylogenetically distinct WOR types. Two model gut microbes containing six types of WOR and Tup were shown to assimilate W. Two of the WORs were natively purified and found to contain W. The enzymes catalyzed the conversion of toxic aldehydes to the corresponding acid, with one WOR carrying out an electron bifurcation reaction coupling aldehyde oxidation to the simultaneous reduction of NAD(+) and of the redox protein ferredoxin. Such aldehydes are present in cooked foods and are produced as antimicrobials by gut microbiome metabolism. This aldehyde detoxification strategy is dependent on the availability of W to the microbe. The functions of other WORs in the gut microbiome that do not oxidize aldehydes remain unknown. W is generally beyond detection (

The genome of the archaeon Pyrobaculum aerophilum (Topt ~ 100 °C) contains an operon (PAE2859–2861) encoding a putative pyranopterin-containing oxidoreductase of unknown function and metal content. These genes (with one gene modified to encode a His-affinity tag) were inserted into the fermentative anaerobic archaeon, Pyrococcus furiosus (Topt ~ 100 °C). Dye-linked assays of cytoplasmic extracts from recombinant P. furiosus show that the P. aerophilum enzyme is a thiosulfate reductase (Tsr) and reduces thiosulfate but not polysulfide. The enzyme (Tsr–Mo) from molybdenum-grown cells contains Mo (Mo:W = 9:1) while the enzyme (Tsr–W) from tungsten-grown cells contains mainly W (Mo:W = 1:6). Purified Tsr–Mo has over ten times the activity (Vmax = 1580 vs. 141 µmol min−1 mg−1) and twice the affinity for thiosulfate (Km = ~ 100 vs. ~ 200 μM) than Tsr–W and is reduced at a lower potential (Epeak = − 255 vs − 402 mV). Tsr–Mo and Tsr–W proteins are heterodimers lacking the membrane anchor subunit (PAE2861). Recombinant P. furiosus expressing P. aerophilum Tsr could not use thiosulfate as a terminal electron acceptor. P. furiosus contains five pyranopterin-containing enzymes, all of which utilize W. P. aerophilum Tsr–Mo is the first example of an active Mo-containing enzyme produced in P. furiosus.

Electron bifurcation plays a key role in anaerobic energy metabolism, but it is a relatively new discovery, and only limited mechanistic information is available on the diverse enzymes that employ it. Herein, we focused on the bifurcating electron transfer flavoprotein (ETF) from the hyperthermophilic archaeon Pyrobaculum aerophilum. The EtfABCX enzyme complex couples NADH oxidation to the endergonic reduction of ferredoxin and exergonic reduction of menaquinone. We developed a model for the enzyme structure by using nondenaturing MS, cross-linking, and homology modeling in which EtfA, -B, and -C each contained FAD, whereas EtfX contained two [4Fe-4S] clusters. On the basis of analyses using transient absorption, EPR, and optical titrations with NADH or inorganic reductants with and without NAD(+), we propose a catalytic cycle involving formation of an intermediary NAD(+)-bound complex. A charge transfer signal revealed an intriguing interplay of flavin semiquinones and a protein conformational change that gated electron transfer between the low- and high-potential pathways. We found that despite a common bifurcating flavin site, the proposed EtfABCX catalytic cycle is distinct from that of the genetically unrelated bifurcating NADH-dependent ferredoxin NADP(+) oxidoreductase (NfnI). The two enzymes particularly differed in the role of NAD(+), the resting and bifurcating-ready states of the enzymes, how electron flow is gated, and the two two-electron cycles constituting the overall four-electron reaction. We conclude that P. aerophilum EtfABCX provides a model catalytic mechanism that builds on and extends previous studies of related bifurcating ETFs and can be applied to the large bifurcating ETF family.

The electron-transferring flavoprotein-menaquinone oxidoreductase ABCX (EtfABCX), also known as FixABCX for its role in nitrogen-fixing organisms, is a member of a family of electron-transferring flavoproteins that catalyze electron bifurcation. EtfABCX enables endergonic reduction of ferredoxin (E°′ ∼−450 mV) using NADH (E°′ −320 mV) as the electron donor by coupling this reaction to the exergonic reduction of menaquinone (E°′ −80 mV). Here we report the 2.9 Å structure of EtfABCX, a membrane-associated flavin-based electron bifurcation (FBEB) complex, from a thermophilic bacterium. EtfABCX forms a superdimer with two membrane-associated EtfCs at the dimer interface that contain two bound menaquinones. The structure reveals that, in contrast to previous predictions, the low-potential electrons bifurcated from EtfAB are most likely directly transferred to ferredoxin, while high-potential electrons reduce the quinone via two [4Fe-4S] clusters in EtfX. Surprisingly, EtfX shares remarkable structural similarity with mammalian [4Fe-4S] cluster-containing ETF ubiquinone oxidoreductase (ETF-QO), suggesting an unexpected evolutionary link between bifurcating and nonbifurcating systems. Based on this structure and spectroscopic studies of a closely related EtfABCX, we propose a detailed mechanism of the catalytic cycle and the accompanying structural changes in this membrane-associated FBEB system.

Modern day aerobic respiration in mitochondria involving complex I converts redox energy into chemical energy and likely evolved from a simple anaerobic system now represented by hydrogen gas-evolving hydrogenase (MBH) where protons are the terminal electron acceptor. Here we present the cryo-EM structure of an early ancestor in the evolution of complex I, the elemental sulfur (S0)-reducing reductase MBS. Three highly conserved protein loops linking cytoplasmic and membrane domains enable scalable energy conversion in all three complexes. MBS contains two proton pumps compared to one in MBH and likely conserves twice the energy. The structure also reveals evolutionary adaptations of MBH that enabled S0 reduction by MBS catalyzed by a site-differentiated iron-sulfur cluster without participation of protons or amino acid residues. This is the simplest mechanism proposed for reduction of inorganic or organic disulfides. It is of fundamental significance in the iron and sulfur-rich volcanic environments of early earth and possibly the origin of life. MBS provides a new perspective on the evolution of modern-day respiratory complexes and of catalysis by biological iron-sulfur clusters., The sulfur-reducing enzyme MBS and the hydrogen-gas evolving MBH are the evolutionary link between the ancestor Mrp antiporter and the mitochondrial respiratory complex I. Here, the authors characterise MBS from the hyperthermophilic archaeon Pyrococcus furiosus, solve its cryo-EM structure and discuss the structural evolution from Mrp to MBH and MBS and to the modern-day complex I.

Arsenate is a notorious toxicant that is known to disrupt multiple biochemical pathways. Many microorganisms have developed mechanisms to detoxify arsenate using the ArsC-type arsenate reductase, and some even use arsenate as a terminal electron acceptor for respiration involving arsenate respiratory reductase (Arr). ArsC-type reductases have been studied extensively, but the phylogenetically unrelated Arr system is less investigated and has not been characterized from Archaea. Here, we heterologously expressed the genes encoding Arr from the crenarchaeon Pyrobaculum aerophilum in the euryarchaeon Pyrococcus furiosus, both of which grow optimally near 100°C. Recombinant P. furiosus was grown on molybdenum (Mo)- or tungsten (W)-containing medium, and two types of recombinant Arr enzymes were purified, one containing Mo (Arr-Mo) and one containing W (Arr-W). Purified Arr-Mo had a 140-fold higher specific activity in arsenate [As(V)] reduction than Arr-W, and Arr-Mo also reduced arsenite [As(III)]. The P. furiosus strain expressing Arr-Mo (the Arr strain) was able to use arsenate as a terminal electron acceptor during growth on peptides. In addition, the Arr strain had increased tolerance compared to that of the parent strain to arsenate and also, surprisingly, to arsenite. Compared to the parent, the Arr strain accumulated intracellularly almost an order of magnitude more arsenic when cells were grown in the presence of arsenite. X-ray absorption spectroscopy (XAS) results suggest that the Arr strain of P. furiosus improves its tolerance to arsenite by increasing production of less-toxic arsenate and nontoxic methylated arsenicals compared to that by the parent. IMPORTANCE Arsenate respiratory reductases (Arr) are much less characterized than the detoxifying arsenate reductase system. The heterologous expression and characterization of an Arr from Pyrobaculum aerophilum in Pyrococcus furiosus provides new insights into the function of this enzyme. From in vivo studies, production of Arr not only enabled P. furiosus to use arsenate [As(V)] as a terminal electron acceptor, it also provided the organism with a higher resistance to arsenate and also, surprisingly, to arsenite [As(III)]. In contrast to the tungsten-containing oxidoreductase enzymes natively produced by P. furiosus, recombinant P. aerophilum Arr was much more active with molybdenum than with tungsten. It is also, to our knowledge, the only characterized Arr to be active with both molybdenum and tungsten in the active site.

The NADPH-dependent cytoplasmic [NiFe]-hydrogenase (SHI) from the hyperthermophile Pyrococcus furiosus, which grows optimally near 100°C, is extremely thermostable and has many in vitro applications, including cofactor generation and hydrogen production. In particular, SHI is used in a cell-free synthetic pathway that contains more than a dozen other enzymes and produces three times more hydrogen (12 H2/glucose) from sugars compared to cellular fermentations (4 H2/glucose). We previously reported homologous over-expression and rapid purification of an affinity-tagged (9x-His) version of SHI, which is a heterotetrameric enzyme. However, about 30% of the enzyme that was purified contained an inactive trimeric form of SHI lacking the catalytic [NiFe]-containing subunit. Herein, we constructed a strain of P. furiosus that contained a second set of the eight genes involved in the maturation of the catalytic subunit and insertion of the [NiFe]-site, along with a second set of the four genes encoding the SHI structural subunits. This resulted in a 40% higher yield of the purified affinity-tagged enzyme and the content of the inactive trimeric form decreased to 5% of the total protein. These results bode well for the future production of active SHI for both basic and applied purposes.

Electron bifurcation has recently gained acceptance as the third mechanism of energy conservation in which energy is conserved through the coupling of exergonic and endergonic reactions. A structure-based mechanism of bifurcation has been elucidated recently for the flavin-based enzyme NADH-dependent ferredoxin NADP+ oxidoreductase I (NfnI) from the hyperthermophillic archaeon Pyrococcus furiosus. NfnI is thought to be involved in maintaining the cellular redox balance, producing NADPH for biosynthesis by recycling the two other primary redox carriers, NADH and ferredoxin. The P. furiosus genome encodes an NfnI paralog termed NfnII, and the two are differentially expressed, depending on the growth conditions. In this study, we show that deletion of the genes encoding either NfnI or NfnII affects the cellular concentrations of NAD(P)H and particularly NADPH. This results in a moderate to severe growth phenotype in deletion mutants, demonstrating a key role for each enzyme in maintaining redox homeostasis. Despite their similarity in primary sequence and cofactor content, crystallographic, kinetic, and mass spectrometry analyses reveal that there are fundamental structural differences between the two enzymes, and NfnII does not catalyze the NfnI bifurcating reaction. Instead, it exhibits non-bifurcating ferredoxin NADP oxidoreductase-type activity. NfnII is therefore proposed to be a bifunctional enzyme and also to catalyze a bifurcating reaction, although its third substrate, in addition to ferredoxin and NADP(H), is as yet unknown.

Flavin-based electron transfer bifurcation is emerging as a fundamental and powerful mechanism for conservation and deployment of electrochemical energy in enzymatic systems. In this process, a pair of electrons is acquired at intermediate reduction potential (i.e. intermediate reducing power), and each electron is passed to a different acceptor, one with lower and the other with higher reducing power, leading to “bifurcation.” It is believed that a strongly reducing semiquinone species is essential for this process, and it is expected that this species should be kinetically short-lived. We now demonstrate that the presence of a short-lived anionic flavin semiquinone (ASQ) is not sufficient to infer the existence of bifurcating activity, although such a species may be necessary for the process. We have used transient absorption spectroscopy to compare the rates and mechanisms of decay of ASQ generated photochemically in bifurcating NADH-dependent ferredoxin-NADP+ oxidoreductase and the non-bifurcating flavoproteins nitroreductase, NADH oxidase, and flavodoxin. We found that different mechanisms dominate ASQ decay in the different protein environments, producing lifetimes ranging over 2 orders of magnitude. Capacity for electron transfer among redox cofactors versus charge recombination with nearby donors can explain the range of ASQ lifetimes that we observe. Our results support a model wherein efficient electron propagation can explain the short lifetime of the ASQ of bifurcating NADH-dependent ferredoxin-NADP+ oxidoreductase I and can be an indication of capacity for electron bifurcation.

Caldicellulosiruptor bescii is an extremely thermophilic, cellulolytic bacterium with a growth optimum at 78 °C and is the most thermophilic cellulose degrader known. It is an attractive target for biotechnological applications, but metabolic engineering will require an in-depth understanding of its primary pathways. A previous analysis of its genome uncovered evidence that C. bescii may have a completely uncharacterized aspect to its redox metabolism, involving a tungsten-containing oxidoreductase of unknown function. Herein, we purified and characterized this new member of the aldehyde ferredoxin oxidoreductase family of tungstoenzymes. We show that it is a heterodimeric glyceraldehyde-3-phosphate (GAP) ferredoxin oxidoreductase (GOR) present not only in all known Caldicellulosiruptor species, but also in 44 mostly anaerobic bacterial genera. GOR is phylogenetically distinct from the monomeric GAP-oxidizing enzyme found previously in several Archaea. We found that its large subunit (GOR-L) contains a single tungstopterin site and one iron-sulfur [4Fe-4S] cluster, that the small subunit (GOR-S) contains four [4Fe-4S] clusters, and that GOR uses ferredoxin as an electron acceptor. Deletion of either subunit resulted in a distinct growth phenotype on both C(5) and C(6) sugars, with an increased lag phase, but higher cell densities. Using metabolomics and kinetic analyses, we show that GOR functions in parallel with the conventional GAP dehydrogenase, providing an alternative ferredoxin-dependent glycolytic pathway. These two pathways likely facilitate the recycling of reduced redox carriers (NADH and ferredoxin) in response to environmental H(2) concentrations. This metabolic flexibility has important implications for the future engineering of this and related species.

Hydrogen production by water splitting energized by biomass sugars is one of the most promising technologies for distributed green H2 production. Direct H2 generation from NADPH, catalysed by an NADPH-dependent, soluble [NiFe]-hydrogenase (SH1) is thermodynamically unfavourable, resulting in slow volumetric productivity. We designed the biomimetic electron transport chain from NADPH to H2 by the introduction of an oxygen-insensitive electron mediator benzyl viologen (BV) and an enzyme (NADPH rubredoxin oxidoreductase, NROR), catalysing electron transport between NADPH and BV. The H2 generation rates using this biomimetic chain increased by approximately five-fold compared to those catalysed only by SH1. The peak volumetric H2 productivity via the in vitro enzymatic pathway comprised of hyperthermophilic glucose 6-phosphate dehydrogenase, 6-phosphogluconolactonase, and 6-phosphogluconate dehydrogenase, NROR, and SH1 was 310 mmol H2/L h−1, the highest rate yet reported. The concept of biomimetic electron transport chains could be applied to both in vitro and in vivo H2 production biosystems and artificial photosynthesis.

Acetyl-Coenzyme A carboxylase (ACC), malonyl-CoA reductase (MCR), and malonic semialdehyde reductase (MRS) convert HCO3- and acetyl-CoA into 3-hydroxypropionate (3HP) in the 3-hydroxypropionate/4-hydroxybutyrate carbon fixation cycle resident in the extremely thermoacidophilic archaeon Metallosphaera sedula. These three enzymes, when introduced into the hyperthermophilic archaeon Pyrococcus furiosus, enable production of 3HP from maltose and CO2 . Sub-optimal function of ACC was hypothesized to be limiting for production of 3HP, so accessory enzymes carbonic anhydrase (CA) and biotin protein ligase (BPL) from M. sedula were produced recombinantly in Escherichia coli to assess their function. P. furiosus lacks a native, functional CA, while the M. sedula CA (Msed_0390) has a specific activity comparable to other microbial versions of this enzyme. M. sedula BPL (Msed_2010) was shown to biotinylate the β-subunit (biotin carboxyl carrier protein) of the ACC in vitro. Since the native BPLs in E. coli and P. furiosus may not adequately biotinylate the M. sedula ACC, the carboxylase was produced in P. furiosus by co-expression with the M. sedula BPL. The baseline production strain, containing only the ACC, MCR, and MSR, grown in a CO2 -sparged bioreactor reached titers of approximately 40 mg/L 3HP. Strains in which either the CA or BPL accessory enzyme from M. sedula was added to the pathway resulted in improved titers, 120 or 370 mg/L, respectively. The addition of both M. sedula CA and BPL, however, yielded intermediate titers of 3HP (240 mg/L), indicating that the effects of CA and BPL on the engineered 3HP pathway were not additive, possible reasons for which are discussed. While further efforts to improve 3HP production by regulating gene dosage, improving carbon flux and optimizing bioreactor operation are needed, these results illustrate the ancillary benefits of accessory enzymes for incorporating CO2 into 3HP production in metabolically engineered P. furiosus, and hint at the important role that CA and BPL likely play in the native 3HP/4HB pathway in M. sedula. Biotechnol. Bioeng. 2016;113: 2652-2660. © 2016 Wiley Periodicals, Inc.

The hyperthermophilic archaeon, Pyrococcus furiosus, grows optimally near 100°C by fermenting sugars to acetate, carbon dioxide and molecular hydrogen as the major end products. The organism has recently been exploited to produce biofuels using a temperature-dependent metabolic switch using genes from microorganisms that grow near 70°C. However, little is known about its metabolism at the lower temperatures. We show here that P. furiosus produces acetoin (3-hydroxybutanone) as a major product at temperatures below 80°C. A novel type of acetolactate synthase (ALS), which is involved in branched-chain amino acid biosynthesis, is responsible and deletion of the als gene abolishes acetoin production. Accordingly, deletion of als in a strain of P. furiosus containing a novel pathway for ethanol production significantly improved the yield of ethanol. These results also demonstrate that P. furiosus is a potential platform for the biological production of acetoin at temperatures in the 70-80°C range.

Electron bifurcating, [FeFe]-hydrogenases are recently described members of the hydrogenase family and catalyze a combination of exergonic and endergonic electron exchanges between three carriers (2 ferredoxinred− + NAD(P)H + 3 H+ = 2 ferredoxinox + NAD(P)+ + 2 H2). A thermodynamic analysis of the bifurcating, [FeFe]-hydrogenase reaction, using electron path-independent variables, quantified potential biological roles of the reaction without requiring enzyme details. The bifurcating [FeFe]-hydrogenase reaction, like all bifurcating reactions, can be written as a sum of two non-bifurcating reactions. Therefore, the thermodynamic properties of the bifurcating reaction can never exceed the properties of the individual, non-bifurcating, reactions. The bifurcating [FeFe]-hydrogenase reaction has three competitive properties: 1) enabling NAD(P)H-driven proton reduction at pH2 higher than the concurrent operation of the two, non-bifurcating reactions, 2) oxidation of NAD(P)H and ferredoxin simultaneously in a 1:1 ratio, both are produced during typical glucose fermentations, and 3) enhanced energy conservation (~10 kJ mol−1 H2) relative to concurrent operation of the two, non-bifurcating reactions. Our analysis demonstrated ferredoxin E°′ largely determines the sensitivity of the bifurcating reaction to pH2, modulation of the reduced/oxidized electron carrier ratios contributed less to equilibria shifts. Hydrogenase thermodynamics data were integrated with typical and non-typical glycolysis pathways to evaluate achieving the ‘Thauer limit’ (4 H2 per glucose) as a function of temperature and pH2. For instance, the bifurcating [FeFe]-hydrogenase reaction permits the Thauer limit at 60 °C if pH 2 ≤ ~10 mbar. The results also predict Archaea, expressing a non-typical glycolysis pathway, would not benefit from a bifurcating [FeFe]-hydrogenase reaction; interestingly, no Archaea have been observed experimentally with a [FeFe]-hydrogenase enzyme.

Caldicellulosiruptor bescii is an extremely thermophilic cellulolytic bacterium with great potential for consolidated bioprocessing of renewable plant biomass. Since it does not natively produce ethanol, metabolic engineering is required to create strains with this capability. Previous efforts involved the heterologous expression of the gene encoding a bifunctional alcohol dehydrogenase, AdhE, which uses NADH as the electron donor to reduce acetyl-CoA to ethanol. Acetyl-CoA produced from sugar oxidation also generates reduced ferredoxin but there is no known pathway for the transfer of electrons from reduced ferredoxin to NAD in C. bescii. Herein, we engineered a strain of C. bescii using a more stable genetic background than previously reported and heterologously-expressed adhE from Clostridium thermocellum (which grows optimally (Topt) at 60 °C) with and without co-expression of the membrane-bound Rnf complex from Thermoanaerobacter sp. X514 (Topt 60 °C). Rnf is an energy-conserving, reduced ferredoxin NAD oxidoreductase encoded by six genes (rnfCDGEAB). It was produced in a catalytically active form in C. bescii that utilized the largest DNA construct to be expressed in this organism. The new genetic lineage containing AdhE resulted in increased ethanol production compared to previous reports. Ethanol production was further enhanced by the presence of Rnf, which also resulted in decreased production of pyruvate, acetoin and an uncharacterized compound as unwanted side-products. Using crystalline cellulose as the growth substrate for the Rnf-containing strain, 75 mM (3.5 g/L) ethanol was produced at 60 °C, which is 5-fold higher than that reported previously. This underlines the importance of redox balancing and paves the way for achieving even higher ethanol titers in C. bescii. Keywords: Biofuel, Consolidated bioprocessing, Ferredoxin, Ethanol, Thermophile, C. bescii

Six multidomain glycoside hydrolases (GHs), CelA (Athe_1867), CelB (Athe_1859), CelC (Athe_1857), CelD (Athe_1866), CelE (Athe_1865), and CelF (Athe_1860) are encoded in the Caldicellulosiruptor bescii glucan degradation locus (GDL). Each GH was affinity-tagged, overexpressed, and purified from recombinant C. bescii for side-by-side characterization in vitro and to examine the contribution of each of these enzymes to microcrystalline cellulose hydrolysis in vivo. All six recombinant GDL GHs were glycosylated, and deletion of glycosyltransferase Athe_1864 eliminated this posttranslational modification. A simplex centroid mixture experimental design revealed that in vitro optimal mixtures of the GDL GHs were predominantly CelA, CelC, and CelE, had low to moderate proportions of CelB and CelD, and minimal CelF. The best binary mixture contained CelA + CelB in a 3:2 molar ratio, whereas the best ternary mixture was composed of CelA + CelC + CelE in equimolar amounts. Neither the native C. bescii secretome nor cocktails of GDL GHs in vitro exceeded 25% of cellulose hydrolysis observed for wild-type C. bescii in vivo. C. bescii deletion strains lacking specific GDL GHs could be restored to wild-type degradation levels with the exogenous addition of either 5 µg/ml of recombinant GDL GH cocktails based on the natural secretome or mixtures optimized in vitro. Also, the addition of CelA up to 100 µg/ml provided no significant additional benefit. These results suggest that the C. bescii secretome is naturally balanced to achieve optimal synergy for cellulose degradation. They also reinforce the importance of microbial contributions to microcrystalline cellulose hydrolysis and suggest that mass action effects from glucan fermentation shift equilibria to drive degradation.

To address national and global low-carbon fuel targets, there is great interest in alternative plant species such as cassava (Manihot esculenta), which are high-yielding, resilient, and are easily converted to fuels using the existing technology. In this study the genes encoding hyperthermophilic archaeal starch-hydrolyzing enzymes, α-amylase and amylopullulanase from Pyrococcus furiosus and glucoamylase from Sulfolobus solfataricus, together with the gene encoding a modified ADP-glucose pyrophosphorylase (glgC) from Escherichia coli, were simultaneously expressed in cassava roots to enhance starch accumulation and its subsequent hydrolysis to sugar. A total of 13 multigene expressing transgenic lines were generated and characterized phenotypically and genotypically. Gene expression analysis using quantitative RT-PCR showed that the microbial genes are expressed in the transgenic roots. Multigene-expressing transgenic lines produced up to 60% more storage root yield than the non-transgenic control, likely due to glgC expression. Total protein extracted from the transgenic roots showed up to 10-fold higher starch-degrading activity in vitro than the protein extracted from the non-transgenic control. Interestingly, transgenic tubers released threefold more glucose than the non-transgenic control when incubated at 85°C for 21-h without exogenous application of thermostable enzymes, suggesting that the archaeal enzymes produced in planta maintain their activity and thermostability.

Hydrogenases catalyze the simplest of chemical reactions, the reversible interconversion of protons, electrons, and hydrogen gas. These enzymes have potential to be utilized for several biotechnological applications, such as in vitro hydrogen production from renewable materials and in enzyme-based fuel cells for electricity generation. Based on the metal content of their catalytic sites, hydrogenases are classified as either [NiFe], [FeFe], or mononuclear Fe enzymes, and [NiFe] hydrogenases are further categorized into five groups based on the sequences of the catalytic subunits. This chapter describes recombinant engineering strategies, purification procedures, and catalytic properties of two distinct types of [NiFe] hydrogenase from Pyrococcus furiosus, a microorganism with an optimal growth temperature of 100°C. These enzymes are termed soluble hydrogenase I (SHI, group 3) and membrane-bound hydrogenase (MBH, group 4). The two hydrogenases were affinity-tagged to facilitate their purification and the purified enzymes have been used for biochemical, mechanistic, and structural analyses. The results have provided us with new insights into how catalysis by SHI is achieved, which could also lead to the development of catalysts for economic hydrogen production, and knowledge of how MBH couples hydrogen gas production to conservation of energy in the form of an ion gradient. The methods described in this chapter provide the basis for these studies.

Caldicellulosiruptor bescii grows optimally at 78°C and is able to decompose high concentrations of lignocellulosic plant biomass without the need for thermochemical pretreatment. C. bescii ferments both C 5 and C 6 sugars primarily to hydrogen gas, lactate, acetate, and CO 2 and is of particular interest for metabolic engineering applications given the recent availability of a genetic system. Developing optimal strains for technological use requires a detailed understanding of primary metabolism, particularly when the goal is to divert all available reductant (electrons) toward highly reduced products such as biofuels. During an analysis of the C. bescii genome sequence for oxidoreductase-type enzymes, evidence was uncovered to suggest that the primary redox metabolism of C. bescii has a completely uncharacterized aspect involving tungsten, a rarely used element in biology. An active tungsten utilization pathway in C. bescii was demonstrated by the heterologous production of a tungsten-requiring, aldehyde-oxidizing enzyme (AOR) from the hyperthermophilic archaeon Pyrococcus furiosus . Furthermore, C. bescii also contains a tungsten-based AOR-type enzyme, here termed XOR, which is phylogenetically unique, representing a completely new member of the AOR tungstoenzyme family. Moreover, in C. bescii , XOR represents ca. 2% of the cytoplasmic protein. XOR is proposed to play a key, but as yet undetermined, role in the primary redox metabolism of this cellulolytic microorganism.

Metabolically engineered strains of the hyperthermophile Pyrococcus furiosus (Topt 95–100°C), designed to produce 3-hydroxypropionate (3HP) from maltose and CO2 using enzymes from the Metallosphaera sedula (Topt 73°C) carbon fixation cycle, were examined with respect to the impact of heterologous gene expression on metabolic activity, fitness at optimal and sub-optimal temperatures, gas-liquid mass transfer in gas-intensive bioreactors, and potential bottlenecks arising from product formation. Transcriptomic comparisons of wild-type P. furiosus, a genetically-tractable, naturally-competent mutant (COM1), and COM1-based strains engineered for 3HP production revealed numerous differences after being shifted from 95°C to 72°C, where product formation catalyzed by the heterologously-produced M. sedula enzymes occurred. At 72°C, significantly higher levels of metabolic activity and a stress response were evident in 3HP-forming strains compared to the non-producing parent strain (COM1). Gas–liquid mass transfer limitations were apparent, given that 3HP titers and volumetric productivity in stirred bioreactors could be increased over 10-fold by increased agitation and higher CO2 sparging rates, from 18 mg/L to 276 mg/L and from 0.7 mg/L/h to 11 mg/L/h, respectively. 3HP formation triggered transcription of genes for protein stabilization and turnover, RNA degradation, and reactive oxygen species detoxification. The results here support the prospects of using thermally diverse sources of pathways and enzymes in metabolically engineered strains designed for product formation at sub-optimal growth temperatures. Biotechnol. Bioeng. 2015;112: 1533–1543. © 2015 Wiley Periodicals, Inc.

Improving access to the carbohydrate content of lignocellulose is key to reducing recalcitrance for microbial deconstruction and conversion to fuels and chemicals. Caldicellulosiruptor bescii completely solubilizes naked microcrystalline cellulose, yet this transformation is impeded within the context of the plant cell wall by a network of lignin and hemicellulose. Here, the bioavailability of carbohydrates to C. bescii at 70°C was examined for reduced lignin transgenic switchgrass lines COMT3(+) and MYB Trans, their corresponding parental lines (cultivar Alamo) COMT3(−) and MYB wild type (WT), and the natural variant cultivar Cave-in-Rock (CR). Transgenic modification improved carbohydrate solubilization by C. bescii to 15% (2.3-fold) for MYB and to 36% (1.5-fold) for COMT, comparable to the levels achieved for the natural variant, CR (36%). Carbohydrate solubilization was nearly doubled after two consecutive microbial fermentations compared to one microbial step, but it never exceeded 50% overall. Hydrothermal treatment (180°C) prior to microbial steps improved solubilization 3.7-fold for the most recalcitrant line (MYB WT) and increased carbohydrate recovery to nearly 50% for the least recalcitrant lines [COMT3(+) and CR]. Alternating microbial and hydrothermal steps (T→M→T→M) further increased bioavailability, achieving carbohydrate solubilization ranging from 50% for MYB WT to above 70% for COMT3(+) and CR. Incomplete carbohydrate solubilization suggests that cellulose in the highly lignified residue was inaccessible; indeed, residue from the T→M→T→M treatment was primarily glucan and inert materials (lignin and ash). While C. bescii could significantly solubilize the transgenic switchgrass lines and natural variant tested here, additional or alternative strategies (physical, chemical, enzymatic, and/or genetic) are needed to eliminate recalcitrance. IMPORTANCE Key to a microbial process for solubilization of plant biomass is the organism's access to the carbohydrate content of lignocellulose. Economically viable routes will characteristically minimize physical, chemical, and biological pretreatment such that microbial steps contribute to the greatest extent possible. Recently, transgenic versions of plants and trees have been developed with the intention of lowering the barrier to lignocellulose conversion, with particular focus on lignin content and composition. Here, the extremely thermophilic bacterium Caldicellulosiruptor bescii was used to solubilize natural and genetically modified switchgrass lines, with and without the aid of hydrothermal treatment. For lignocellulose conversion, it is clear that the microorganism, plant biomass substrate, and processing steps must all be considered simultaneously to achieve optimal results. Whether switchgrass lines engineered for low lignin or natural variants with desirable properties are used, conversion will depend on microbial access to crystalline cellulose in the plant cell wall.

The first generation of biochemical studies of complex, iron-sulfur-cluster-containing [FeFe]-hydrogenases and Mo-nitrogenase were carried out on enzymes purified from Clostridium pasteurianum (strain W5). These Previous studies suggested that two distinct [FeFe]-hydrogenases are expressed differentially under nitrogen-fixing and non-nitrogen-fixing conditions. As a result, the first characterized [FeFe]-hydrogenase (CpI) is presumed to have a primary role in central metabolism, recycling reduced electron carriers that accumulate during fermentation via proton reduction. A role for capturing reducing equivalents released as hydrogen during nitrogen fixation has been proposed for the second hydrogenase, CpII. Biochemical characterization of CpI and CpII indicated CpI has extremely high hydrogen production activity in comparison to CpII, while CpII has elevated hydrogen oxidation activity in comparison to CpI when assayed under the same conditions. This suggests that these enzymes have evolved a catalytic bias to support their respective physiological functions. Using the published genome of C. pasteurianum (strain W5) hydrogenase sequences were identified, including the already known [NiFe]-hydrogenase, CpI, and CpII sequences, and a third hydrogenase, CpIII was identified in the genome as well.We identified four hydrogenases: three [FeFe]-hydrogenases, one of which had not previously been known, and one [NiFe]-hydrogenase in the published genome of C. pasteurianum (strain W5). Quantitative real-time PCR experiments were performed in order to analyze transcript abundance of the hydrogenases under diazotrophic and non diazatrophic growthconditions. show There is a markedly reduced levels of CpI gene expression together with concomitant increases in CpII gene expression under nitrogen-fixing conditions. Structure-based analyses of the CpI and CpII sequences reveals variations in their catalytic sites that may contribute to their alternative physiological roles. This work demonstrates that the physiological roles of CpI and CpII are to evolve and to consume hydrogen, respectively, in concurrence with their catalytic activities in vitro, with CpII capturing excess reducing equivalents under nitrogen fixation conditions. Comparison of the primary sequences of CpI and CpII and their homologs provides an initial basis for identifying key structural determinants that modulate hydrogen production and hydrogen oxidation activities.

Recent investigations into ferredoxin-dependent transhydrogenases, a class of enzymes responsible for electron transport, have highlighted the biological importance of flavin-based electron bifurcation (FBEB). FBEB generates biomolecules with very low reduction potential by coupling the oxidation of an electron donor with intermediate potential to the reduction of high and low potential molecules. Bifurcating systems can generate biomolecules with very low reduction potentials, such as reduced ferredoxin (Fd), from species such as NADPH. Metabolic systems that use bifurcation are more efficient and confer a competitive advantage for the organisms that harbor them. Structural models are now available for two NADH-dependent ferredoxin-NADP+ oxidoreductase (Nfn) complexes. These models, together with spectroscopic studies, have provided considerable insight into the catalytic process of FBEB. However, much about the mechanism and regulation of these multi-subunit proteins remains unclear. Using hydrogen/deuterium exchange mass spectrometry (HDX-MS) and statistical coupling analysis (SCA), we identified specific pathways of communication within the model FBEB system, Nfn from Pyrococus furiosus, under conditions at each step of the catalytic cycle. HDX-MS revealed evidence for allosteric coupling across protein subunits upon nucleotide and ferredoxin binding. SCA uncovered a network of co-evolving residues that can provide connectivity across the complex. Together, the HDX-MS and SCA data show that protein allostery occurs across the ensemble of iron‑sulfur cofactors and ligand binding sites using specific pathways that connect domains allowing them to function as dynamically coordinated units.

Electron transport phosphorylation (ETP), one of three known biochemical mechanisms for conserving energy, couples oxidation-reduction reactions to the translocation of ions across cell membranes (Table 1). Although we tend to associate ETP with high potential acceptors such as oxygen, it also comes into play using low potential acceptors. In such cases, energy can accumulate through sequential translocation of ions across cellular membranes through low-energy- yielding reactions.

The heterotrophic, hyperthermophilic archaeon Pyrococcus furiosus is a new addition to the growing list of genetically-tractable microorganisms suitable for metabolic engineering to produce liquid fuels and industrial chemicals. P. furiosus was recently engineered to generate 3-hydroxypropionate (3-HP) from CO 2 and acetyl-CoA by the heterologous-expression of three enzymes from the CO 2 fixation cycle of the thermoacidophilic archaeon Metallosphaera sedula using a thermally-triggered induction system. The acetyl-CoA for this pathway is generated from glucose catabolism that in wild-type P. furiosus is converted to acetate with concurrent ATP production by the heterotetrameric (α 2 β 2 ) acetyl-CoA synthetase (ACS). Hence ACS in the engineered 3-HP production strain (MW56) competes with the heterologous pathway for acetyl-CoA. Herein we show that strains of MW56 lacking the α-subunit of either of the two ACSs previously characterized from P. furiosus (ACSI and ACSII) exhibit a three-fold increase in specific 3-HP production. The ΔACSIα strain displayed only a minor defect in growth on either maltose or peptides, while no growth defect on these substrates was observed with the ΔACSIIα strain. Deletion of individual and multiple ACS subunits was also shown to decrease CoA release activity for several different CoA ester substrates in addition to acetyl-CoA, information that will be extremely useful for future metabolic engineering endeavors in P. furiosus .

The hyperthermophilic archaeonPyrococcus furiosusgrows by fermenting peptides and carbohydrates to organic acids. In the terminal step, acyl-CoA synthetase (ACS) isoenzymes convert acyl-CoA derivatives to the corresponding acid and conserve energy in the form of ATP. ACS1 and ACS2 were previously purified fromP. furiosusand haveα2β2structures but the genome contains genes encoding three additionalα-subunits. The ten possible combinations ofαandβgenes were expressed inE. coliand each resulted in stable and activeα2β2isoenzymes. Theα-subunit of each isoenzyme determined CoA-based substrate specificity and between them they accounted for the CoA derivatives of fourteen amino acids. Theβ-subunit determined preference for adenine or guanine nucleotides. The GTP-generating isoenzymes are proposed to play a role in gluconeogenesis by producing GTP for GTP-dependent phosphoenolpyruvate carboxykinase and for other GTP-dependent processes. Transcriptional and proteomic data showed that all ten isoenzymes are constitutively expressed indicating that both ATP and GTP are generated from the metabolism of most of the amino acids. A phylogenetic analysis showed that the ACSs ofP. furiosusand other members of the Thermococcales are evolutionarily distinct from those found throughout the rest of biology, including those of other hyperthermophilic archaea.

) is the most abundant pentose in ourplanet.Itisamajorbuildingblockofhemicellulose,oneofthemain constituents of lignocellulosic biomass. Xylose com-prises ca. 20–30% weight of plant matter. Because it is noteconomically feasible to separate xylose from other biomasssugars, the co-utilization of pentose and hexose is essential tothe success of the production of biocommodities frombiomass. In principle, one mole of xylose when combinedwith H

Reverse gyrase is an enzyme that induces positive supercoiling in closed circular DNA in vitro. It is unique to thermophilic organisms and found without exception in all microorganisms defined as hyperthermophiles, that is, those having optimal growth temperatures of 80 °C and above. Although its in vivo role has not been clearly defined, it has been implicated in stabilizing DNA at high temperatures. Whether or not it is absolutely required for growth at these high temperatures has yet to be fully determined. In a previous study with an organism that has an optimal growth temperature of 85 °C, it was shown that the enzyme is not a prerequisite for life at extreme temperatures as disruption of its gene did not result in a lethal phenotype at the supraoptimal growth temperature of 90 °C. Herein we show that the enzyme is absolutely required for microbial growth at 95 °C, which in this case is a suboptimal growth temperature. Deletion of the gene encoding the reverse gyrase of the model hyperthermophilic archaeon Pyrococcus furiosus, which has an optimal growth temperature of 100 °C, revealed that the gene is required for growth at 95 °C, as well as at 100 °C. The results suggest that a temperature threshold above 90 °C exists, wherein the activity of reverse gyrase is absolutely necessary to maintain a correct DNA twist for any organism growing at such temperature extremes.

Iron–sulfur clusters are ubiquitous in biology and function in electron transfer and catalysis. They are assembled from iron and cysteine sulfur on protein scaffolds. Iron is typically stored as iron oxyhydroxide, ferrihydrite, encapsulated in 12 nm shells of ferritin, which buffers cellular iron availability. Here we have characterized IssA, a protein that stores iron and sulfur as thioferrate, an inorganic anionic polymer previously unknown in biology. IssA forms nanoparticles reaching 300 nm in diameter and is the largest natural metalloprotein complex known. It is a member of a widely distributed protein family that includes nitrogenase maturation factors, NifB and NifX. IssA nanoparticles are visible by electron microscopy as electron-dense bodies in the cytoplasm. Purified nanoparticles appear to be generated from 20 nm units containing ∼6,400 Fe atoms and ∼170 IssA monomers. In support of roles in both iron–sulfur storage and cluster biosynthesis, IssA reconstitutes the [4Fe-4S] cluster in ferredoxin in vitro., The biosynthesis of iron-sulfur clusters in anaerobic organisms has not been extensively investigated. Here, the authors identify and characterize a multi-subunit protein that stores iron and sulfur in thioferrate for the assembly of the clusters in Pyrococcus furiosus.

C-1 carriers are essential cofactors in all domains of life, and in Archaea, these can be derivatives of tetrahydromethanopterin (H(4)-MPT) or tetrahydrofolate (H(4)-folate). Their synthesis requires 6-hydroxymethyl-7,8-dihydropterin diphosphate (6-HMDP) as the precursor, but the nature of pathways that lead to its formation were unknown until the recent discovery of the GTP cyclohydrolase IB/MptA family that catalyzes the first step, the conversion of GTP to dihydroneopterin 2',3'-cyclic phosphate or 7,8-dihydroneopterin triphosphate [El Yacoubi, B.; et al. (2006) J. Biol. Chem., 281, 37586-37593 and Grochowski, L. L.; et al. (2007) Biochemistry46, 6658-6667]. Using a combination of comparative genomics analyses, heterologous complementation tests, and in vitro assays, we show that the archaeal protein families COG2098 and COG1634 specify two of the missing 6-HMDP synthesis enzymes. Members of the COG2098 family catalyze the formation of 6-hydroxymethyl-7,8-dihydropterin from 7,8-dihydroneopterin, while members of the COG1634 family catalyze the formation of 6-HMDP from 6-hydroxymethyl-7,8-dihydropterin. The discovery of these missing genes solves a long-standing mystery and provides novel examples of convergent evolutions where proteins of dissimilar architectures perform the same biochemical function.

Crenarchaeotal genomes encode the 3-hydroxypropionate/4-hydroxybutyrate (3-HP/4-HB) cycle for carbon dioxide fixation. Of the 13 enzymes putatively comprising the cycle, several of them, including methylmalonyl-coenzyme A (CoA) epimerase (MCE) and methylmalonyl-CoA mutase (MCM), which convert ( S )-methylmalonyl-CoA to succinyl-CoA, have not been confirmed and characterized biochemically. In the genome of Metallosphaera sedula (optimal temperature [ T opt ], 73°C), the gene encoding MCE (Msed_0639) is adjacent to that encoding the catalytic subunit of MCM-α (Msed_0638), while the gene for the coenzyme B 12 -binding subunit of MCM (MCM-β) is located remotely (Msed_2055). The expression of all three genes was significantly upregulated under autotrophic compared to heterotrophic growth conditions, implying a role in CO 2 fixation. Recombinant forms of MCE and MCM were produced in Escherichia coli ; soluble, active MCM was produced only if MCM-α and MCM-β were coexpressed. MCE is a homodimer and MCM is a heterotetramer (α 2 β 2 ) with specific activities of 218 and 2.2 μmol/min/mg, respectively, at 75°C. The heterotetrameric MCM differs from the homo- or heterodimeric orthologs in other organisms. MCE was activated by divalent cations (Ni 2+ , Co 2+ , and Mg 2+ ), and the predicted metal binding/active sites were identified through sequence alignments with less-thermophilic MCEs. The conserved coenzyme B 12 -binding motif ( DXHXXG -SXL-GG) was identified in M. sedula MCM-β. The two enzymes together catalyzed the two-step conversion of ( S )-methylmalonyl-CoA to succinyl-CoA, consistent with their proposed role in the 3-HP/4-HB cycle. Based on the highly conserved occurrence of single copies of MCE and MCM in Sulfolobaceae genomes, the M. sedula enzymes are likely to be representatives of these enzymes in the 3-HP/4-HB cycle in crenarchaeal thermoacidophiles.

Transcriptional and enzymatic analyses of Pyrococcus furiosus previously indicated that three proteins play key roles in the metabolism of elemental sulfur (S 0 ): a membrane-bound oxidoreductase complex (MBX), a cytoplasmic coenzyme A-dependent NADPH sulfur oxidoreductase (NSR), and sulfur-induced protein A (SipA). Deletion strains, referred to as MBX1, NSR1, and SIP1, respectively, have now been constructed by homologous recombination utilizing the uracil auxotrophic COM1 parent strain (Δ pyrF ). The growth of all three mutants on maltose was comparable without S 0 , but in its presence, the growth of MBX1 was greatly impaired while the growth of NSR1 and SIP1 was largely unaffected. In the presence of S 0 , MBX1 produced little, if any, sulfide but much more acetate (per unit of protein) than the parent strain, demonstrating that MBX plays a critical role in S 0 reduction and energy conservation. In contrast, comparable amounts of sulfide and acetate were produced by NSR1 and the parent strain, indicating that NSR is not essential for energy conservation during S 0 reduction. Differences in transcriptional responses to S 0 in NSR1 suggest that two sulfide dehydrogenase isoenzymes provide a compensatory NADPH-dependent S 0 reduction system. Genes controlled by the S 0 -responsive regulator SurR were not as highly regulated in MBX1 and NSR1. SIP1 produced the same amount of acetate but more sulfide than the parent strain. That SipA is not essential for growth on S 0 indicates that it is not required for detoxification of metal sulfides, as previously suggested. A model is proposed for S 0 reduction by P. furiosus with roles for MBX and NSR in bioenergetics and for SipA in iron-sulfur metabolism.

Virtually all cellular processes are carried out by dynamic molecular assemblies or multiprotein complexes, the compositions of which are largely undefined. They cannot be predicted solely from bioinformatics analyses nor are there well defined techniques currently available to unequivocally identify protein complexes (PCs). To address this issue, we attempted to directly determine the identity of PCs from native microbial biomass using Pyrococcus furiosus, a hyperthermophilic archaeon that grows optimally at 100 degrees C, as the model organism. Novel PCs were identified by large scale fractionation of the native proteome using non-denaturing, sequential column chromatography under anaerobic, reducing conditions. A total of 967 distinct P. furiosus proteins were identified by mass spectrometry (nano LC-ESI-MS/MS), representing approximately 80% of the cytoplasmic proteins. Based on the co-fractionation of proteins that are encoded by adjacent genes on the chromosome, 106 potential heteromeric PCs containing 243 proteins were identified, only 20 of which were known or expected. In addition to those of unknown function, novel and uncharacterized PCs were identified that are proposed to be involved in the metabolism of amino acids (10), carbohydrates (four), lipids (two), vitamins and metals (three), and DNA and RNA (nine). A further 30 potential PCs were classified as tentative, and the remaining potential PCs (13) were classified as weakly interacting. Some major advantages of native biomass fractionation for PC identification are that it provides a road map for the (partial) purification of native forms of novel and uncharacterized PCs, and the results can be utilized for the recombinant production of low abundance PCs to provide enough material for detailed structural and biochemical analyses.

Key enzymes involved in end-product formation were identified in Thermoanaerobacterium saccharolyticum JW/SL-YS485, a thermophilic anaerobic bacterium under consideration as a biological catalyst for the conversion of cellulosic biomass to ethanol. Based on enzymatic assays and genome sequence analyses, pathways were identified that would lead to the generation of all major products from xylose fermentation: lactate, acetate, ethanol, hydrogen, and carbon dioxide. Pyruvate ferredoxin oxidoreductase is the primary pyruvate decarboxylating enzyme, producing carbon dioxide, reduced ferredoxin, and acetyl-CoA, and ferredoxin is likely oxidized by a specific hydrogenase. It is concluded that enzymes are present in this organism that could theoretically produce ethanol from carbohydrates at high yield.

The myristoylated calcium sensor SOS3 and its interacting protein kinase, SOS2, play critical regulatory roles in salt tolerance. Mutations in either of these proteins render Arabidopsis thaliana plants hypersensitive to salt stress. We report here the isolation and characterization of a mutant called enh1-1 that enhances the salt sensitivity of sos3-1 and also causes increased salt sensitivity by itself. ENH1 encodes a chloroplast-localized protein with a PDZ domain at the N-terminal region and a rubredoxin domain in the C-terminal part. Rubredoxins are known to be involved in the reduction of superoxide in some anaerobic bacteria. The enh1-1 mutation causes enhanced accumulation of reactive oxygen species (ROS), particularly under salt stress. ROS also accumulate to higher levels in sos2-1 but not in sos3-1 mutants. The enh1-1 mutation does not enhance sos2-1 phenotypes. Also, enh1-1 and sos2-1 mutants, but not sos3-1 mutants, show increased sensitivity to oxidative stress. These results indicate that ENH1 functions in the detoxification of reactive oxygen species resulting from salt stress by participating in a new salt tolerance pathway that may involve SOS2 but not SOS3.

The hypothetical protein PF0899 is a 95-residue peptide from the hyperthermophilic archaeon Pyrococcus furiosus that represents a gene family with six members. P. furiosus ORF PF0899 has been cloned, expressed and crystallized and its structure has been determined by the Southeast Collaboratory for Structural Genomics (http://www.secsg.org). The structure was solved using the SCA2Structure pipeline from multiple data sets and has been refined to 1.85 A against the highest resolution data set collected (a presumed gold derivative), with a crystallographic R factor of 21.0% and R(free) of 24.0%. The refined structure shows some structural similarity to a wedge-shaped domain observed in the structure of the major capsid protein from bacteriophage HK97, suggesting that PF0899 may be a structural protein.

An early divergence in evolution has resulted in two prokaryotic domains, the Bacteria and the Archaea. Whereas the central metabolic routes of bacteria and eukaryotes are generally well-conserved, variant pathways have developed in Archaea involving several novel enzymes with a distinct control. A spectacular example of convergent evolution concerns the glucose-degrading pathways of saccharolytic archaea. The identification, characterization and comparison of the glycolytic enzymes of a variety of phylogenetic lineages have revealed a mosaic of canonical and novel enzymes in the archaeal variants of the Embden–Meyerhof and the Entner–Doudoroff pathways. By means of integrating results from biochemical and genetic studies with recently obtained comparative and functional genomics data, the structure and function of the archaeal glycolytic routes, the participating enzymes and their regulation are re-evaluated.

Marine hydrothermal microorganisms respond rapidly to changes in the concentrations and availability of metals within their environment. Hyperthermophilic archaea appear to possess novel mechanisms for metal detoxification, dissimilatory metal reduction and metal assimilation that may be absent in their mesophilic and bacterial counterparts. For example, tungsten was found in high concentrations in a hydrothermal sulfide deposit where hyperthermophiles were also most abundant, consistent with the unique requirement of these organisms for this element. Furthermore, newly isolated genera of iron-reducing hyperthermophiles expand the scope of carbon cycling in hydrothermal environments. The advent of genome sequences and new molecular techniques will facilitate our further understanding of microbe–mineral interactions in these environments.

Pyrococcus furiosus grows optimally near 100°C using peptides and carbohydrates as carbon sources, and it reduces elemental sulfur (S 0 ), if present, to H 2 S. Tungsten (W), an element rarely used in biology, is required for optimal growth, and three different tungsten-containing enzymes have been previously purified from this organism. They all oxidize aldehydes of various types and are thought to play primary roles in the catabolism of sugars or amino acids. Here, the purification of a fourth tungsten-containing enzyme, termed WOR 4, from cell extracts of P. furiosus grown with S 0 is described. This was achieved by monitoring through multiple chromatography steps the W that is not associated with the three characterized tungstoenzymes. The N-terminal sequence of WOR 4 and the approximate molecular weight of its subunit determined electrophoretically (69,000) correspond to the product of an ORF (PF1961, wor4 ) present in the complete genome sequence of P. furiosus . WOR 4 is a homodimer and contains approximately one W, three Fe, three or four acid-labile sulfide, and one Ca atom per subunit. The visible and electron paramagnetic resonance spectra of the oxidized and reduced enzyme indicate the presence of an unusual iron-sulfur chromophore. WOR 4 does not oxidize aliphatic or aromatic aldehydes or hydroxy acids, nor does it reduce keto acids. Consistent with prior microarray data, the protein could not be purified from P. furiosus cells grown in the absence of S 0 , suggesting that it may have a role in S 0 metabolism.

It was recently proposed that anaerobic microorganisms contain a new pathway for detoxification of reactive oxygen species. This is centered around a novel mononuclear iron-containing enzyme, superoxide reductase (SOR), which catalyzes the reduction, rather than the dismutation, of superoxide to hydrogen peroxide. A surprisingly large amount of relevant data has accumulated in the two years or so since the proposal was made. Herein we address the questions: to what extent has the pathway been validated, and what fundamental issues have yet to be answered in considering the response of anaerobes to reactive oxygen species? The evidence for superoxide reduction by SOR is now overwhelming and comes from a variety of anaerobic and microaerophilic species. Moreover, the available spectroscopic and structural information provide a convincing case that the catalytic Fe site of SOR is structurally and electronically tuned to mediate superoxide reduction rather than oxidation. Kinetic analyses also support the original proposal of NAD(P)H, via rubredoxin and NAD(P)H:rubredoxin oxidoreductase, as the source of reductant. What is still to be determined is the fate of the peroxide generated by the SOR reaction. In particular, the role of otherwise well-characterized proteins like rubrerythrin, NADH peroxidase, and rubredoxin:oxygen oxidoreductase in "anaerobic" oxygen metabolism has yet to be established.

Although as many as half of all proteins are thought to require a metal cofactor, the metalloproteomes of microorganisms remain relatively unexplored. Microorganisms from different environments are likely to vary greatly in the metals that they assimilate, not just among the metals with well-characterized roles but also those lacking any known function. Herein we investigated the metal utilization of two microorganisms that were isolated from very similar environments and are of interest because of potential roles in the immobilization of heavy metals, such as uranium and chromium. The metals assimilated and their concentrations in the cytoplasm of Desulfovibrio vulgaris strain Hildenborough (DvH) and Enterobacter cloacae strain Hanford (EcH) varied dramatically, with a larger number of metals present in Enterobacter. For example, a total of 9 and 19 metals were assimilated into their cytoplasmic fractions, respectively, and DvH did not assimilate significant amounts of zinc or copper whereas EcH assimilated both. However, bioinformatic analysis of their genome sequences revealed a comparable number of predicted metalloproteins, 813 in DvH and 953 in EcH. These allowed some rationalization of the types of metal assimilated in some cases (Fe, Cu, Mo, W, V) but not in others (Zn, Nd, Ce, Pr, Dy, Hf and Th). It was also shown that U binds an unknown soluble protein in EcH but this incorporation was the result of extracellular U binding to cytoplasmic components after cell lysis.

A number of unique microorganisms that thrive at temperatures of 90°C or higher have been isolated in the last 2 decades. One of the best studied of these so-called hyperthermophiles is the anaerobic archaeon Pyrococcus furiosus, which grows optimally at 100°C by the fermentation of carbohydrates and peptides (15). The organism uses a modified Embden-Meyerhof glycolytic pathway that contains several novel enzymes (13, 23). For example, both hexokinase and phosphofructokinase are ADP- rather than ATP-dependent enzymes (24, 48). In addition, the expected glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase enzymes, which would convert glyceraldehyde-3-phosphate to 3-phosphoglycerate with the concomitant phosphorylation of ADP to ATP and reduction of NAD+ to NADH, appear to be replaced by a single enzyme, glyceraldehyde-3-phosphate:ferredoxin oxidoreductase (GAPOR). GAPOR oxidizes glyceraldehyde-3-phosphate directly to 3-phosphoglycerate and uses ferredoxin rather than NAD+ as the electron acceptor. Purified GAPOR is a unique tungsten-containing enzyme that has no known analog in mesophilic archaea or bacteria (31). Another unusual step in glucose catabolism includes the conversion of acetyl coenzyme A (acetyl-CoA) to acetate. In anaerobic bacteria, this is a two-step process via acetyl phosphate catalyzed by phosphotransacetylase and acetate kinase. In P. furiosus, however, these two enzymes are replaced by acetyl-CoA synthetase, which converts acetyl-CoA directly to acetate and phosphorylates ADP to ATP (29). Other enzymes involved in glucose oxidation that have been purified from Pyrococcus species include pyruvate:ferredoxin oxidoreductase (4), enolase (36), and triosephosphate isomerase (25). In contrast to the enzymes described previously, these are quite similar (except in thermostability) to their mesophilic counterparts. P. furiosus also synthesizes glucose during growth on peptide-derived amino acids (15), but less work has been done to explore the gluconeogenic pathway. This is thought to occur by a reversal of the conventional Embden-Meyerhof pathway, and the activities of the enzymes proposed to be involved have been measured in cell extracts (44). Of the gluconeogenic-specific enzymes, only phosphoglycerate kinase (19) has been purified from Pyrococcus species. Herein we focus on the enzyme that carries out the first step in the conversion of pyruvate to glucose, phosphoenolpyruvate (PEP) synthetase, which catalyzes the phosphorylation of pyruvate according to equation 1: 1 Transcriptional analyses indicated that the cellular concentration of PEP synthetase increased when a glycolytic substrate such as maltose was added to the growth medium of P. furiosus (38, 39). To explain this result, it was suggested that this enzyme functions in glycolysis (reverse of equation 1) as well as in gluconeogenesis (39). However, there are no kinetic data to support this contention. It was, therefore, of some relevance to determine the catalytic properties of PEP synthetase from this organism. Surprisingly, little is known about PEP synthetases from prokaryotes, in spite of their importance in controlling carbon flow during glucose metabolism (20, 28, 37, 45, 47). The only one to be studied extensively is that from Escherichia coli (10, 11, 32, 33, 34). It is a cofactorless homodimer with a molecular mass of 150 kDa and catalyzes the phosphorylation of pyruvate by using ATP to generate PEP, AMP, and phosphate. Only two other PEP synthetases have been purified and examined to any extent, and both are from archaea, the moderately thermophilic methanogen Methanobacterium thermoautotrophicum (14) and the heterotrophic hyperthermophile Staphylothermus marinus (8, 9, 16, 17). The quaternary structure of the former enzyme was not reported, but S. marinus PEP synthetase exists as a large homomultimeric complex containing 24 subunits (17). Conversely, while the kinetic properties of the latter enzyme are unknown, those of the M. thermoautotrophicum enzyme are similar to those of E. coli PEP synthetase (14). It was therefore of some interest to investigate the physical and catalytic properties of the enzyme from P. furiosus, especially as the enzyme has been reported to be sensitive to inactivation by oxygen (44). For example, did the enzyme exist as a large complex, did it function in both gluconeogenesis and glycolysis, did it utilize nucleotides other than ATP as the phosphate donor for PEP synthesis, and what may be the cause of its oxygen sensitivity?

The gene ( sipA ) encoding the sulfur-induced protein A (PF2025) is highly upregulated during growth of Pyrococcus furiosus on elemental sulfur (S 0 ). Expression of sipA is regulated by sulfide, the product of S 0 reduction, but in an iron-dependent manner. SipA is proposed to play a role in intracellular iron sulfide detoxification.

The electronic and redox properties of the iron-sulfur cluster and tungsten center in the as-isolated and sulfide-activated forms of formaldehyde ferredoxin oxidoreductase (FOR) from Thermococcus litoralis (Tl) have been investigated by using the combination of EPR and variable-temperature magnetic circular dichroism (VTMCD) spectroscopies. The results reveal a [Fe4S4]2+,+ cluster (Em=-368mV) that undergoes redox cycling between an oxidized form with an S=0 ground state and a reduced form that exists as a pH- and medium-dependent mixture of S=3/2 (g=5.4; E/D=0.33) and S=1/2 (g=2.03, 1.93, 1.86) ground states, with the former dominating in the presence of 50% (v/v) glycerol. Three distinct types of W(V) EPR signals have been observed during dye-mediated redox titration of as-isolated Tl FOR. The initial resonance observed upon oxidation, termed the "low-potential" W(V) species (g=1.977, 1.898, 1.843), corresponds to approximately 25-30% of the total W and undergoes redox cycling between W(IV)/ W(V) and W(V)/W(VI) states at physiologically relevant potentials (Em= -335 and -280 mV, respectively). At higher potentials a minor "mid-potential" W(V) species, g= 1.983, 1.956, 1.932, accounting for less than 5 % of the total W, appears with a midpoint potential of -34 mV and persists up to at least + 300 mV. At potentials above 0 mV, a major "high-potential" W(V) signal, g= 1.981, 1.956, 1.883, accounting for 30-40% of the total W, appears at a midpoint potential of +184 mV. As-isolated samples of Tl FOR were found to undergo an approximately 8-fold enhancement in activity on incubation with excess Na2S under reducing conditions and the sulfide-activated Tl FOR was partially inactivated by cyanide. The spectroscopic and redox properties of the sulfide-activated Tl FOR are quite distinct from those of the as-isolated enzyme, with loss of the low-potential species and changes in both the mid-potential W(V) species (g= 1.981, 1.950, 1.931; Em = -265 mV) and high-potential W(V) species (g=1.981, 1.952, 1.895; Em = +65 mV). Taken together, the W(V) species in sulfide-activated samples of Tl FOR maximally account for only 15% of the total W. Both types of high-potential W(V) species were lost upon incubation with cyanide and the sulfide-activated high-potential species is converted into the as-isolated high-potential species upon exposure to air. Structural models are proposed for each of the observed W(V) species and both types of mid-potential and high-potential species are proposed to be artifacts of ligand-based oxidation of W(VI) species. A W(VI) species with terminal sulfido or thiol ligands is proposed to be responsible for the catalytic activity in sulfide-activated samples of Tl FOR.