Discovery, structure and mechanism of a tetraether lipid synthase.

Archaea synthesize isoprenoid-based ether-linked membrane lipids, which enable them to withstand extreme environmental conditions, such as high temperatures, high salinity, and low or high pH values1-5. In some archaea, such as Methanocaldococcus jannaschii, these lipids are further modified by forming carbon-carbon bonds between the termini of two lipid tails within one glycerophospholipid to generate the macrocyclic archaeol or forming two carbon-carbon bonds between the termini of two lipid tails from two glycerophospholipids to generate the macrocycle glycerol dibiphytanyl glycerol tetraether (GDGT)1,2. GDGT contains two 40-carbon lipid chains (biphytanyl chains) that span both leaflets of the membrane, providing enhanced stability to extreme conditions. How these specialized lipids are formed has puzzled scientists for decades. The reaction necessitates the coupling of two completely inert sp3-hybridized carbon centres, which, to our knowledge, has not been observed in nature. Here we show that the gene product of mj0619 from M. jannaschii, which encodes a radical S-adenosylmethionine enzyme, is responsible for biphytanyl chain formation during synthesis of both the macrocyclic archaeol and GDGT membrane lipids6. Structures of the enzyme show the presence of four metallocofactors: three [Fe4S4] clusters and one mononuclear rubredoxin-like iron ion. In vitro mechanistic studies show that Csp3-Csp3 bond formation takes place on fully saturated archaeal lipid substrates and involves an intermediate bond between the substrate carbon and a sulfur of one of the [Fe4S4] clusters. Our results not only establish the biosynthetic route for tetraether formation but also improve the use of GDGT in GDGT-based paleoclimatology indices7-10.

A Membrane-Associated DHH-DHHA1 Nuclease Degrades Type III CRISPR Second Messenger.

Type III CRISPR-Cas systems initiate an intracellular signaling pathway to confer immunity. The signaling pathway includes synthesis of cyclic oligo-adenylate (cOA) and activation of the RNase activity of type III accessory ribonuclease Csm6/Csx1 by cOA. After the immune response, cOA should be cleared on time to avoid constant cellular RNA degradation. In this study, we find a metal-dependent cOA degradation activity in Sulfolobus islandicus. The activity is associated with the cell membrane and able to accelerate cOA clearance at a high cOA level. Further, we show that a metal-dependent and membrane-associated DHH-DHHA1 family nuclease (MAD) rapidly cleaves cOA and deactivates Csx1 ribonuclease. The cOA degradation efficiency of MAD is much higher than the cellular ring nuclease. However, the subcellular organization may prevent it from degrading nascent cOA. Together, the data suggest that MAD acts as the second cOA degrader after the ring nuclease to remove diffused redundant cOA.

MALDI-TOF analysis of a novel extremophile peptide purified from Halarchaeum acidiphilum ASDL78 with antiarchaeal and antibacterial activities.

In hypersaline environments, halophilic archaea synthesize antimicrobial substances called halocins. There is a promise to make new drugs for antibiotic-resistant strains. Here, we report the antibacterial activity of a new haloarchaea selected from Lut Desert, Iran. A total of 38 isolated halophilic bacteria and archaea were screened for the antagonistic activity test of each strain against other bacterial and archaeal strains. Finally, a strain, recognized as Halarchaeum acidiphilum, with a fast grown strain and high antagonistic potential against different strains was identified by morphological, physiological, and molecular characteristics. The halocin was produced in a semisolid submerge medium and partially purified by heat treatments and molecular weight ultrafiltration cutoff (3, 50, and 10 kDa). It was a cell-free, heat-resistant (85°C for 2 h) protein with a molecular mass near to 20 kDa produced at the endpoint of logarithmic growth. The molecular weight of halocin was 17 kDa, and indicated no apparent homology with known halocins, suggesting that this might be a new halocin. Therefore, a new strain belonging to Halarchaeum genus was isolated and characterized here that produced an antimicrobial and anti-haloarchaea halocin.

Complete sequential assignment and secondary structure prediction of the cannulae forming protein CanA from the hyperthermophilic archaeon Pyrodictium abyssi.

CanA from Pyrodictium abyssi forms a heat-resistant organic hollow-fiber network together with CanB and CanC. An N-terminally truncated construct of CanA (K1-CanA) gave NMR spectra of good quality that could be assigned by three-dimensional NMR methods on 15N and 13C-15N enriched protein. We assigned the chemical shifts of 96% of all backbone 1HN atoms, 98% of all backbone 15N atoms, 100% of all 13Cα atoms, 100% of all 1Hα atoms, 90% of all 13C' atoms, and 100% of the 13Cß atoms. Two short helices and 10 ß-strands are estimated from an analysis of the chemical shifts leading to a secondary structure content of K1-CanA of 6% helices, 44% ß-pleated sheets, and 50% coils.

Characterization of Extracellular Protease from the Haloarcheon Halococcus sp. Strain GUGFAWS-3 (MF425611).

Halococcus agarilyticus GUGFAWS-3 (MF425611) was isolated from a marine white sponge of Haliclona sp., inhabiting the rocks in the intertidal region of Anjuna, Goa, India. Uniquely, the microbe simultaneously produces two halo-extremozymes in 25% NaCl, namely protease and lipase at 49.5 ± 0.4 and 3.67 ± 0.02 (U mL-1), respectively. The protease is constitutively produced in starch mineral salts medium with consistent 4 ± 1.0 mm zone of enzyme production, regardless of the non-availability of protein as substrate. The ethanol precipitated enzyme on dialysis and Sephadex G-200 gel filtration chromatography was partially purified to 12.26-fold and was active between 20 and 80 °C, 0-5 M NaCl, and pH 3-13. Optimum activity, however, was at 70 °C, 3 M NaCl, and pH 7. The enzyme was thermo stable at 70 °C with 50.26 ± 2.40% of relative enzyme activity at 75 min. Furthermore, it was stable in the presence of polar and non-polar organic solvents, detergents, and hydrocarbons. Several metal cations enhanced its activity in the order of Ca2+ > Ni2+ > Fe3+ > Co2+ > Mg2+ > Cu2+ > Mn2+. Dependence of enzyme on cysteine; serine, and metal ions was confirmed by ß-mercaptoethanol; PMSF and EDTA, respectively which induced its partial inhibition. Additionally, protease inhibited in vitro biofilm formation in Staphylococcus aureus. Conclusively, the production of a neutral halo-thermophilic protease is reported for the first time in the genus Halococcus.

FruBPase II and ADP-PFK1 are involved in the modulation of carbon flow in the metabolism of carbohydrates in Methanosarcina acetivorans.

To enhance our understanding of the control of archaeal carbon central metabolism, a detailed analysis of the regulation mechanisms of both fructose1,6-bisphosphatase (FruBPase) and ADP-phosphofructokinase-1 (ADP-PFK1) was carried out in the methanogen Methanosarcina acetivorans. No correlations were found among the transcript levels of the MA_1152 and MA_3563 (frubpase type II and pfk1) genes, the FruBPase and ADP-PFK1 activities, and their protein contents. The kinetics of the recombinant FruBPase II and ADP-PFK1 were hyperbolic and showed simple mixed-type inhibition by AMP and ATP, respectively. Under physiological metabolite concentrations, the FruBPase II and ADP-PFK1 activities were strongly modulated by their inhibitors. To assess whether these enzymes were also regulated by a phosphorylation/dephosphorylation process, the recombinant enzymes and cytosolic-enriched fractions were incubated in the presence of commercial protein phosphatase or protein kinase. De-phosphorylation of ADP-PFK1 slightly decreased its activity (i.e. Vmax) and did not change its kinetic parameters and oligomeric state. Thus, the data indicated a predominant metabolic regulation of both FruBPase and ADP-PFK1 activities by adenine nucleotides and suggested high degrees of control on the respective pathway fluxes.

TYW1: A Radical SAM Enzyme Involved in the Biosynthesis of Wybutosine Bases.

Transfer RNA is extensively modified by the actions of a variety of enzymes. The radical S-adenosyl-l-methionine enzyme TYW1 modifies tRNAPhe forming the characteristic tricyclic ring via the condensation of carbons 2 and 3 of pyruvate. This chapter details methods that are required for studies of TYW1.

Radical S-Adenosyl-l-Methionine (SAM) Enzyme Involved in the Maturation of the Nitrogenase Cluster.

Nitrogenase is the only known enzymatic system that converts atmospheric dinitrogen (N2) into bioavailable ammonia (NH3). The active-site cofactor responsible for this reactivity is a [(R-homocitrate)MoFe7S9C] cluster that is designated as the M-cluster. This important cofactor is assembled stepwise from a pair of [Fe4S4] clusters that become fused into a [Fe8S9C] core before additional refinements take place to complete the biosynthesis. NifB, a member of the radical S-adenosyl-l-methionine (SAM) superfamily, facilitates the conversion of the [Fe4S4] clusters (called the K-cluster) to the [Fe8S9C] core (called the L-cluster). This transformation includes a SAM-dependent carbide insertion with concomitant incorporation of an additional sulfur. While difficulties with the purification of NifB have historically prevented detailed biochemical analyses, we have developed a heterologous expression system in Escherichia coli that yields stable NifB proteins from various N2-fixing methanogenic organisms that can be used for studies. This chapter details the procedures necessary to prepare an active NifB protein. The methods used for the biochemical characterization of the SAM-dependent carbide insertion reactions are also described.

Methods for Studying the Radical SAM Enzymes in Diphthamide Biosynthesis.

Diphthamide is a unique posttranslational modification on translation elongation factor 2 (EF2) in archaea and eukaryotes. Biosynthesis of diphthamide was proposed to involve four steps. The first step is a CC bond forming reaction catalyzed by unique radical S-adenosylmethionine (SAM) enzymes. Classical radical SAM enzymes use SAM and [4Fe-4S] clusters to generate a 5'-deoxyadenynal radical and catalyze numerous reactions. Radical SAM enzymes in diphthamide biosynthesis cleave a different CS bond in SAM to generate a 3-amino-3-carboxypropyl radical and modify a histidine residue of substrate protein EF2. Here, we describe our investigations on these unique radical SAM enzymes, including the preparation, characterization, and activity assays we have developed.

Identification of the Radical SAM Enzymes Involved in the Biosynthesis of Methanopterin and Coenzyme F420 in Methanogens.

Methanogenic archaea represent a source of unique and fascinating anaerobic biochemistry that includes the involvement of many radical S-adenosyl-l-methionine (SAM) enzymes, some of which have well-established functions, while the majority have currently unknown or only partially understood functions. Here, we describe our strategy for the identification of the radical SAM enzyme that catalyzes the two methylation reactions in methanopterin biosynthesis in Methanocaldococcus jannaschii. Additionally, we describe the similar strategy carried out for the identification of the two radical SAM enzymes required for the biosynthesis of the 7,8-didemethyl-8-hydroxy-5-deazariboflavin (F0) moiety of coenzyme F420 in M. jannaschii. This approach can be employed for future functional identification of radical SAM enzymes with currently unknown functions.

The RadA Recombinase and Paralogs of the Hyperthermophilic Archaeon Sulfolobus solfataricus.

Repair of DNA double-strand breaks is a critical function shared by organisms in all three domains of life. The majority of mechanistic understanding of this process has come from characterization of bacterial and eukaryotic proteins, while significantly less is known about analogous activities in the third, archaeal domain. Despite the physical resemblance of archaea to bacteria, archaeal proteins involved in break repair are remarkably similar to those used by eukaryotes. Investigating the function of the archaeal version of these proteins is, in many cases, simpler than working with eukaryotic homologs owing to their robust nature and ease of purification. In this chapter, we describe methods for purification and activity analysis for the RadA recombinase and its paralogs from the hyperthermophilic acidophilic archaeon Sulfolobus solfataricus.

Archaeal Lsm rings as stable self-assembling tectons for protein nanofabrication.

We have exploited the self-assembling properties of archaeal-derived protein Lsmα to generate new supramolecular forms based on its stable ring-shaped heptamer. We show that engineered ring tectons incorporating cysteine sidechains on obverse faces of the Lsmα7 toroid are capable of forming paired and stacked formations. A Cys-modified construct, N10C/E61C-Lsmα, appears to organize into disulfide-mediated tube formations up to 45 nm in length. We additionally report fabrication of cage-like protein clusters through conjugation of Cu2+ to His-tagged variants of the Lsmα7 tecton. These 400 kDa protein capsules are seen as cube particles with visible pores, and are reversibly dissembled into their component ring tectons by EDTA. The ß-rich Lsmα supramolecular assemblies described are amenable to further fusion modifications, or for surface attachment, so providing potential for future applications that exploit the RNA-binding capacity of Lsm proteins, such as sensing applications.

Using the entrapment sequence method as a standard to evaluate key steps of proteomics data analysis process.

BACKGROUND: The mass spectrometry based technical pipeline has provided a high-throughput, high-sensitivity and high-resolution platform for post-genomic biology. Varied models and algorithms are implemented by different tools to improve proteomics data analysis. The target-decoy searching strategy has become the most popular strategy to control false identification in peptide and protein identifications. While this strategy can estimate the false discovery rate (FDR) within a dataset, it cannot directly evaluate the false positive matches in target identifications. RESULTS: As a supplement to target-decoy strategy, the entrapment sequence method was introduced to assess the key steps of mass spectrometry data analysis process, database search engines and quality control methods. Using the entrapment sequences as the standard, we evaluated five database search engines for both the origanal scores and reprocessed scores, as well as four quality control methods in term of quantity and quality aspects. Our results showed that the latest developed search engine MS-GF+ and percolator-embeded quality control method PepDistiller performed best in all tools respectively. Combined with efficient quality control methods, the search engines can improve the low sensitivity of their original scores. Moreover, based on the entrapment sequence method, we proved that filtering the identifications separately could increase the number of identified peptides while improving the confidence level. CONCLUSION: In this study, we have proved that the entrapment sequence method could be an useful strategy to assess the key steps of the mass spectrometry data analysis process. Its applications can be extended to all steps of the common workflow, such as the protein assembling methods and data integration methods.

Secretory production of designed multipeptides displayed on a thermostable bacterial thioredoxin scaffold in Pichia pastoris.

Internal grafting of designed peptides to scaffold proteins is a valuable strategy for a variety of applications including recombinant peptide antigen construction. A peptide epitope from human papillomavirus (HPV) minor capsid protein L2 displayed on thioredoxin (Trx) has been validated preclinically as a broadly protective and low-cost alternative HPV vaccine. Focusing on thioredoxin from the hyperthermophilic archaebacterium Pyrococcus furiosus (PfTrx) as a scaffold, we have constructed a modified Pichia pastoris expression vector and used a PfTrx fusion derivative containing three tandemly repeated copies of a 19 amino acids peptide epitope from HPV-L2 for expression optimization and biochemical-immunological characterization of the Pichia-produced PfTrx-L2 antigen. We show that PfTrx-L2 is produced at high levels (up to 100 mg from a 100 ml starting culture using a multi-cycle induction protocol) and secreted into the culture medium as a highly enriched (>70% pure), non-glycosylated polypeptide that can be purified to homogeneity in a single step. Oxidation and aggregation state, thermal stability and immunogenicity of the endotoxin-free PfTrx-L2 antigen produced in P. pastoris were tested and found to be identical to those of the same antigen produced in Escherichia coli. Secretory production of endotoxin-free PfTrx-peptides in P. pastoris represents a cost- and time-effective alternative to E. coli production. Specifically designed for peptide antigens, the PfTrx-expression vector and conditions described herein are easily transferable to a variety of applications centred on the use of structurally constrained bioactive peptides as immune as well as target-specific binder reagents.

Affinity chromatography of chaperones based on denatured proteins: Analysis of cell lysates of different origin.

Molecular chaperones are involved in folding, oligomerization, transport, and degradation of numerous cellular proteins. Most of chaperones are heat-shock proteins (HSPs). A number of diseases of various organisms are accompanied by changes in the structure and functional activity of chaperones, thereby revealing their vital importance. One of the fundamental properties of chaperones is their ability to bind polypeptides lacking a rigid spatial structure. Here, we demonstrate that affinity chromatography using sorbents with covalently attached denatured proteins allows effective purification and quantitative assessment of their bound protein partners. Using pure Escherichia coli chaperone GroEL (Hsp60), the capacity of denatured pepsin or lysozyme-based affinity sorbents was evaluated as 1 mg and 1.4 mg of GroEL per 1 ml of sorbent, respectively. Cell lysates of bacteria (E. coli, Thermus thermophilus, and Yersinia pseudotuberculosis), archaea (Halorubrum lacusprofundi) as well as the lysate of rat liver mitochondria were analyzed using affinity carrier with denatured lysozyme. It was found that, apart from Hsp60, other proteins with a molecular weight of about 100, 50, 40, and 20 kDa are able to interact with denatured lysozyme.

The identification of a novel Sulfolobus islandicus CAMP-like peptide points to archaeal microorganisms as cell factories for the production of antimicrobial molecules.

BACKGROUND: Pathogenic bacteria easily develop resistance to c onventional antibiotics so that even relatively new molecules are quickly losing efficacy. This strongly encourages the quest of new antimicrobials especially for the treatment of chronic infections. Cationic antimicrobial peptides (CAMPs) are small positively charged peptides with an amphipathic structure, active against Gram-positive and Gram-negative bacteria, fungi, as well as protozoa. RESULTS: A novel (CAMP)-like peptide (VLL-28) was identified in the primary structure of a transcription factor, Stf76, encoded by pSSVx, a hybrid plasmid-virus from the archaeon Sulfolobus islandicus. VLL-28 displays chemical, physical and functional properties typical of CAMPs. Indeed, it has a broad-spectrum antibacterial activity and acquires a defined structure in the presence of membrane mimetics. Furthermore, it exhibits selective leakage and fusogenic capability on vesicles with a lipid composition similar to that of bacterial membranes. VLL-28 localizes not only on the cell membrane but also in the cytoplasm of Escherichia coli and retains the ability to bind nucleic acids. These findings suggest that this CAMP-like peptide could exert its antimicrobial activity both on membrane and intra cellular targets. CONCLUSIONS: VLL-28 is the first CAMP-like peptide identified in the archaeal kingdom, thus pointing to archaeal microorganisms as cell factories to produce antimicrobial molecules of biotechnological interest. Furthermore, results from this work show that DNA/RNA-binding proteins could be used as sources of CAMPs.

Purification and characterization of halo-alkali-thermophilic protease from Halobacterium sp. strain HP25 isolated from raw salt, Lake Qarun, Fayoum, Egypt.

A total of 33 halophilic protease producers were isolated from different salt samples collected from Emisal salt company at Lake Qarun, Fayoum, Egypt. Of these strains, an extremely halophilic strain that grew optimally at 30 % (w/v) NaCl was characterized and identified as Halobacterium sp. strain HP25 based on 16S rRNA gene sequencing and phenotypic characterization. A halo-alkali-thermophilic protease was purified in three successive steps from the culture supernatant. The purified halophilic protease consisted of a single polypeptide chain with a molecular mass of 21 kDa and was enriched 167-fold to a specific activity of 6350 U mg(-1). The purified enzyme was active over a broad pH range from 6.0 to 11.0, with maximum activity at pH 8.0, exhibited a broad temperature range from 30 to 80 °C with optimum activity at 60 °C, and was active at salt concentrations ranging from 5 to 25 % (w/v), with optimum activity at 17 % NaCl (w/v). The K M and V max values of the purified halophilic protease with casein as a substrate were 523 µg mL(-1) and 2500 µg min(-1) mL(-1), respectively. In addition, this enzyme was stable in the tested organic solvents and laundry detergents such methanol, propanol, butanol, hexane, Persil and Ariel. The unusual properties of this enzyme allow it to be used for various applications, such as the ripening of salted fish. Furthermore, its stability and activity in the presence of organic solvents and detergents also allow the use of this enzyme for further novel applications and as an additive in detergent formulations.

Biochemical characterization of a thermostable adenosylmethionine synthetase from the archaeon Pyrococcus furiosus with high catalytic power.

Adenosylmethionine synthetase plays a key role in the biogenesis of the sulfonium compound S-adenosylmethionine, the principal widely used methyl donor in the biological methylations. We report here, for the first time, the characterization of adenosylmethionine synthetase from the hyperthermophilic archaeon Pyrococcus furiosus (PfMAT). The gene PF1866 encoding PfMAT was cloned and expressed, and the recombinant protein was purified to homogeneity. PfMAT shares 51, 63, and 82% sequence identity with the homologous enzymes from Sulfolobus solfataricus, Methanococcus jannaschii, and Thermococcus kodakarensis, respectively. PfMAT is a homodimer of 90 kDa highly thermophilic with an optimum temperature of 90 °C and is characterized by remarkable thermodynamic stability (Tm, 99 °C), kinetic stability, and resistance to guanidine hydrochloride-induced unfolding. The latter process is reversible as demonstrated by the analysis of the refolding process by activity assays and fluorescence measurements. Limited proteolysis experiments indicated that the proteolytic cleavage site is localized at Lys148 and that the C-terminal peptide is necessary for the integrity of the active site. PfMAT shows kinetic features that make it the most efficient catalyst for S-adenosylmethionine synthesis among the characterized MAT from Bacteria and Archaea. Molecular and structural characterization of PfMAT could be useful to improve MAT enzyme engineering for biotechnological applications.

High yield purification of a tagged cytoplasmic [NiFe]-hydrogenase and a catalytically-active nickel-free intermediate form.

The cytoplasmic [NiFe]-hydrogenase I (SHI) of the hyperthermophile Pyrococcus furiosus evolves hydrogen gas (H2) from NADPH. It has been previously used for biohydrogen production from sugars using a mixture of enzymes in an in vitro cell-free synthetic pathway. The theoretical yield (12 H2/glucose) is three times greater than microbial fermentation (4 H2/glucose), making the in vitro approach very promising for large scale biohydrogen production. Further development of this process at an industrial scale is limited by the availability of the H2-producing SHI. To overcome the obstacles of the complex biosynthetic and maturation pathway for the [NiFe] site of SHI, the four gene operon encoding the enzyme was overexpressed in P. furiosus and included a polyhistidine affinity tag. The one-step purification resulted in a 50-fold increase in yield compared to the four-step purification procedure for the native enzyme. A trimeric form was also identified that lacked the [NiFe]-catalytic subunit but catalyzed NADPH oxidation with a specific activity similar to that of the tetrameric form. The presence of an active trimeric intermediate confirms the proposed maturation pathway where, in the terminal step, the NiFe-containing catalytic subunit assembles with NADPH-oxidizing trimeric form to give the active holoenzyme.

Discovery and characterization of the first archaeal dihydromethanopterin reductase, an iron-sulfur flavoprotein from Methanosarcina mazei.

The microbial production of methane by methanogenic archaea is dependent on the synthesis of the pterin-containing cofactor tetrahydromethanopterin (H4MPT). The enzyme catalyzing the last step of H4MPT biosynthesis (dihydromethanopterin reductase) has not previously been identified in methane-producing microorganisms. Previous complementation studies with the methylotrophic bacterium Methylobacterium extorquens have indicated that an uncharacterized archaeal-flavoprotein-like flavoprotein (AfpA) from Methylobacillus flagellatus or Burkholderia xenovorans can replace the activity of a phylogenetically unrelated bacterial dihydromethanopterin reductase (DmrA). We propose that MM1854, a homolog of AfpA from Methanosarcina mazei, catalyzes the last step of H4MPT biosynthesis in methane-producing microorganisms. To test this hypothesis, a six-histidine (His6)-tagged version of MM1854 was produced. Bioinformatic analysis revealed the presence of one flavin mononucleotide (FMN)-binding site and two iron-sulfur cluster sites, consistent with an oxidoreductase enzyme. Purified His6-MM1854 occurred as a homodimer of 29-kDa subunits, and the UV-visible spectrum of the purified protein showed absorbance peaks at 380 and 460 nm, characteristic of oxidized FMN. NAD(P)H was incapable of directly reducing the flavin cofactor, but dithionite eliminated the FMN peaks, indicating successful electron transfer to MM1854. An electron transfer system of NADPH, spinach NADPH-ferredoxin oxidoreductase, and ferredoxin could also reduce the FMN peaks. A newly developed assay indicated that dithiothreitol-reduced MM1854 could transfer electrons to dihydromethanopterin. This assay was also effective with a heat-stable DmrX analog from Methanocaldococcus jannaschii (MJ0208). These results provide the first biochemical evidence that MM1854 and MJ0208 function as archaeal dihydromethanopterin reductases (DmrX) and that ferredoxin may serve as an electron donor.