Four Inserts within the Catalytic Domain Confer Extra Stability and Activity to Hyperthermostable Pyrolysin from Pyrococcus furiosus.

Pyrolysin from the hyperthermophilic archaeon Pyrococcus furiosus is the prototype of the pyrolysin family of the subtilisin-like serine protease superfamily (subtilases). It contains four inserts (IS147, IS29, IS27, and IS8) of unknown function in the catalytic domain. We performed domain deletions and showed that three inserts are either essential (IS147 and IS27) or important (IS8) for efficient maturation of pyrolysin at high temperatures, whereas IS29 is dispensable. The large insert IS147 contains Ca3 and Ca4, two calcium-binding Dx[DN]xDG motifs that are conserved in many pyrolysin-like proteases. Mutagenesis revealed that the Ca3 site contributes to enzyme thermostability and the Ca4 site is necessary for pyrolysin to fold into a maturation-competent conformation. Mature insert-deletion variants were characterized and showed that IS29 and IS8 contribute to enzyme activity and stability, respectively. In the presence of NaCl, pyrolysin undergoes autocleavage at two sites: one within IS29 and the other in IS27 Disrupting the ion pairs in IS27 and IS8 induces autocleavage in the absence of salts. Interestingly, autocleavage products combine noncovalently to form an active, nicked enzyme that is resistant to SDS and urea denaturation. Additionally, a single mutation in IS29 increases resistance to salt-induced autocleavage and further increases enzyme thermostability. Our results suggest that these extra structural elements play a crucial role in adapting pyrolysin to hyperthermal environments.IMPORTANCE Pyrolysin-like proteases belong to the subtilase superfamily and are characterized by large inserts and long C-terminal extensions; however, the role of the inserts in enzyme function is unclear. Our results demonstrate that four inserts in the catalytic domain of hyperthermostable pyrolysin contribute to the folding, maturation, stability, and activity of the enzyme at high temperatures. The modification of extra structural elements in pyrolysin-like proteases is a promising strategy for modulating global structure stability and enzymatic activity of this class of protease.

A pentose bisphosphate pathway for nucleoside degradation in Archaea.

Owing to the absence of the pentose phosphate pathway, the degradation pathway for the ribose moieties of nucleosides is unknown in Archaea. Here, in the archaeon Thermococcus kodakarensis, we identified a metabolic network that links the pentose moieties of nucleosides or nucleotides to central carbon metabolism. The network consists of three nucleoside phosphorylases, an ADP-dependent ribose-1-phosphate kinase and two enzymes of a previously identified NMP degradation pathway, ribose-1,5-bisphosphate isomerase and type III ribulose-1,5-bisphosphate carboxylase/oxygenase. Ribose 1,5-bisphosphate and ribulose 1,5-bisphosphate are intermediates of this pathway, which is thus designated the pentose bisphosphate pathway.

Ionic interactions of Ba2+ blockades in the MthK K+ channel.

The movement and interaction of multiple ions passing through in single file underlie various fundamental K(+) channel properties, from the effective conduction of K(+) ions to channel blockade by Ba(2+) ions. In this study, we used single-channel electrophysiology and x-ray crystallography to probe the interactions of Ba(2+) with permeant ions within the ion conduction pathway of the MthK K(+) channel. We found that, as typical of K(+) channels, the MthK channel was blocked by Ba(2+) at the internal side, and the Ba(2+)-blocking effect was enhanced by external K(+). We also obtained crystal structures of the MthK K(+) channel pore in both Ba(2+)-Na(+) and Ba(2+)-K(+) environments. In the Ba(2+)-Na(+) environment, we found that a single Ba(2+) ion remained bound in the selectivity filter, preferably at site 2, whereas in the Ba(2+)-K(+) environment, Ba(2+) ions were predominantly distributed between sites 3 and 4. These ionic configurations are remarkably consistent with the functional studies and identify a molecular basis for Ba(2+) blockade of K(+) channels.

Ionic strength-dependent conformations of a ubiquitin-like small archaeal modifier protein (SAMP1) from Haloferax volcanii.

Eukaryotic ubiquitin and ubiquitin-like systems play crucial roles in various cellular biological processes. In this work, we determined the solution structure of SAMP1 from Haloferax volcanii by NMR spectroscopy. Under low ionic conditions, SAMP1 presented two distinct conformations, one folded ß-grasp and the other disordered. Interestingly, SAMP1 underwent a conformational conversion from disorder to order with ion concentration increasing, indicating that the ordered conformation is the functional form of SAMP1 under the physiological condition of H. volcanii. Furthermore, SAMP1 could interact with proteasome-activating nucleotidase B, supposing a potential role of SAMP1 in the protein degradation pathway mediated by proteasome.

Effect of organic solvents on the activity and stability of an extracellular protease secreted by the haloalkaliphilic archaeon Natrialba magadii.

The effect of various organic solvents on the activity and stability of an extracellular protease produced by the haloalkaliphilic archaeon Natrialba magadii was tested. This protease was active and stable in aqueous-organic solvent mixtures containing 1.5 M NaCl and glycerol, dimethylsulfoxide (DMSO), N,N-dimethyl formamide, propylenglycol, and dioxane. Among the solvents tested, DMSO, propylenglycol, and glycerol were effective in preserving enzyme stability in suboptimal NaCl concentrations. The stabilizing effect of DMSO on this haloalkaliphilic protease was more efficient at pH 8 than at pH 10, suggesting that DMSO may not substitute for salt to allow halophilic proteins to withstand the effect of high pH values. These results show that Nab. magadii extracellular protease is a solvent tolerant enzyme and suggest a potential application of this haloalkaliphilic protease in aqueous-organic solvent biocatalysis.

Increased stability of malate dehydrogenase from Halobacterium salinarum at low salt concentration in reverse micelles.

The stability of malate dehydrogenase (hMDH) from Halobacterium salinarum in aqueous medium at low salt concentrations (1 and 0.5 M NaCl) was studied at 4 degrees and 25 degrees C. The results showed that hMDH was more stable at the higher salt concentration and the low temperature. hMDH was introduced into reverse micelles of hexadecyltrimethylammonium bromide in cyclohexane with 1-butanol as co-surfactant. The hMDH stability in this system was studied at two omega(0) ([H(2)O]/[surfactant]) values and the effects of salt concentration, presence of substrate and dilution before or after its introduction into reverse micelles were examined. The results showed that the half-life of hMDH dissolved in buffer with 1 M NaCl was 12-50 days in reverse micelles (depending on the various conditions), in contrast to only about 1 day in aqueous medium at 25 degrees C. These observations indicate that reverse micelles provide a microenvironment that allows a much greater stability of this enzyme compared with an aqueous medium.

The search for traces of life: the protective effect of salt on biological macromolecules.

Trapping malate dehydrogenase from the extremely halophilic archaeon Haloarcula marismortui in "dry" salt crystals protects the enzyme against thermal denaturation. Similar protection was not observed for the homologous mesophilic enzyme. In the case of transfer RNA molecules, high salt concentration plays a protective role against thermal degradation allowing activity to be recovered. The results are discussed in the context of exploring the fate of cell-free biological macromolecules in the environment and that of orienting the search for traces of life in planetary exploration.

The thermosome from Methanopyrus kandleri possesses an NH4+-dependent ATPase activity.

The ATPase activity of the thermosome from a methanogen, Methanopyrus kandleri, was characterized in detail. In contrast to all other known chaperonins, enzymatic ATP hydrolysis was found to be strictly dependent on high levels of ammonium salts in vitro. The ths gene encoding the thermosome subunit from the hyperthermophilic M. kandleri was functionally expressed in Escherichia coli and the overproduced polypeptide was assembled into intact thermosome complexes in the mesophilic host. The recombinant particles could be purified by a simple two-step procedure including only one chromatographic step. Structural and biochemical properties of the recombinant protein were closely similar to those of the natural complex. Western blot analysis with an antiserum against the M. kandleri thermosome indicated the presence of at least two subfamilies of archaeal chaperonins.

The effects of salt on the TATA binding protein-DNA interaction from a hyperthermophilic archaeon.

This study investigates the thermodynamics of the interaction of the TATA box binding protein (TBP) from Pyrococcus woesei (Pw) with an oligonucleotide containing a specific binding site. Pw is a hyperthermophilic archeal organism which exists under conditions of high salt and high temperature. A measurable protein-DNA interaction only occurs at high salt concentrations. Isothermal titration calorimetric binding studies were performed under a range of salts (potassium chloride, potassium phosphate, potassium acetate and sodium acetate) at varying concentrations (0.8 to 1.6 M). At the high salt concentrations used the observed equilibrium binding constant increases with increasing salt concentration. This is very different to the effect reported for all other protein-DNA interactions which have been studied at lower salt concentrations. Thermodynamic data suggest that the protein-DNA interaction at high salt concentration is accompanied by the removal of large numbers of water molecules from the buried hydrophobic surface area. In addition, the involvement of ions appears to influence the binding which can be explained by binding of cations in the interface between the electrostatically negative lateral lobes on the protein and the negatively charged DNA.

Identification of cysteine and arginine residues essential for the phosphotransacetylase from Methanosarcina thermophila.

Phosphotransacetylase catalyzes the following reaction: CoASH + CH3CO2PO3(2-) <==> CH3COSCoA + HPO4(2-) (where CoA is coenzyme A). Based on biochemical characterization of the enzyme from the obligate anaerobe Clostridium kluyveri, a ternary mechanism was proposed in which an unspecified cysteine abstracts a proton from CoASH forming a nucleophilic thiolate anion which attacks acetyl phosphate (J. Henkin and R. H. Abeles, Biochemistry 15:3472-3479, 1976). Heterologous production in Escherichia coli of the phosphotransacetylase from Methanosarcina thermophila, an obligately anaerobic methanoarchaeon, allowed site-specific replacements to identify essential residues. All four cysteines present in the sequence were individually replaced with alanine, and the kinetic constants of the altered enzymes were determined. The results indicated that only C159 is essential for activity; however, replacement with serine resulted in a fully active enzyme. Activity of the unaltered phosphotransacetylase was sensitive to N-ethylmaleimide. Inhibition kinetics of altered enzymes indicated that this sensitivity resulted from modification of C312, which is at the active site but itself is nonessential for catalysis. Five arginines were individually replaced with glutamine. Kinetic analysis of the altered enzymes identified R310 as essential for activity. Of the four nonessential for activity, R87 and R133 appear to be involved in binding CoA.