Key amino acid residues conferring enhanced enzyme activity at cold temperatures in an Antarctic polyextremophilic ß-galactosidase.

The Antarctic microorganism Halorubrum lacusprofundi harbors a model polyextremophilic ß-galactosidase that functions in cold, hypersaline conditions. Six amino acid residues potentially important for cold activity were identified by comparative genomics and substituted with evolutionarily conserved residues (N251D, A263S, I299L, F387L, I476V, and V482L) in closely related homologs from mesophilic haloarchaea. Using a homology model, four residues (N251, A263, I299, and F387) were located in the TIM barrel around the active site in domain A, and two residues (I476 and V482) were within coiled or ß-sheet regions in domain B distant to the active site. Site-directed mutagenesis was performed by partial gene synthesis, and enzymes were overproduced from the cold-inducible cspD2 promoter in the genetically tractable Haloarchaeon, Halobacterium sp. NRC-1. Purified enzymes were characterized by steady-state kinetic analysis at temperatures from 0 to 25 °C using the chromogenic substrate o-nitrophenyl-ß-galactoside. All substitutions resulted in altered temperature activity profiles compared with wild type, with five of the six clearly exhibiting reduced catalytic efficiency (kcat/Km) at colder temperatures and/or higher efficiency at warmer temperatures. These results could be accounted for by temperature-dependent changes in both Km and kcat (three substitutions) or either Km or kcat (one substitution each). The effects were correlated with perturbation of charge, hydrogen bonding, or packing, likely affecting the temperature-dependent flexibility and function of the enzyme. Our interdisciplinary approach, incorporating comparative genomics, mutagenesis, enzyme kinetics, and modeling, has shown that divergence of a very small number of amino acid residues can account for the cold temperature function of a polyextremophilic enzyme.

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.

Divalent metal ion-induced folding mechanism of RNase H1 from extreme halophilic archaeon Halobacterium sp. NRC-1.

RNase H1 from Halobacterium sp. NRC-1 (Halo-RNase H1) is characterized by the abundance of acidic residues on the surface, including bi/quad-aspartate site residues. Halo-RNase H1 exists in partially folded (I) and native (N) states in low-salt and high-salt conditions respectively. Its folding is also induced by divalent metal ions. To understand this unique folding mechanism of Halo-RNase H1, the active site mutant (2A-RNase H1), the bi/quad-aspartate site mutant (6A-RNase H1), and the mutant at both sites (8A-RNase H1) were constructed. The far-UV CD spectra of these mutants suggest that 2A-RNase H1 mainly exists in the I state, 6A-RNase H1 exists both in the I and N states, and 8A-RNase H1 mainly exists in the N state in a low salt-condition. These results suggest that folding of Halo-RNase H1 is induced by binding of divalent metal ions to the bi/quad-aspartate site. To examine whether metal-induced folding is unique to Halo-RNase H1, RNase H2 from the same organism (Halo-RNase H2) was overproduced and purified. Halo-RNase H2 exists in the I and N states in low-salt and high-salt conditions respectively, as does Halo-RNase H1. However, this protein exists in the I state even in the presence of divalent metal ions. Halo-RNase H2 exhibits junction ribonuclease activity only in a high-salt condition. A tertiary model of this protein suggests that this protein does not have a quad-aspartate site. We propose that folding of Halo-RNase H1 is induced by binding of divalent metal ion to the quad-aspartate site in a low-salt condition.

Heterologous overexpression, purification and characterisation of an alcohol dehydrogenase (ADH2) from Halobacterium sp. NRC-1.

Replacement of chemical steps with biocatalytic ones is becoming increasingly more interesting due to the remarkable catalytic properties of enzymes, such as their wide range of substrate specificities and variety of chemo-, stereo- and regioselective reactions. This study presents characterisation of an alcohol dehydrogenase (ADH) from the halophilic archaeum Halobacterium sp. NRC-1 (HsADH2). A hexahistidine-tagged recombinant version of HsADH2 (His-HsADH2) was heterologously overexpressed in Haloferax volcanii. The enzyme was purified in one step by immobilised Ni-affinity chromatography. His-HsADH2 was halophilic and mildly thermophilic with optimal activity for ethanol oxidation at 4 M KCl around 60 °C and pH 10.0. The enzyme was extremely stable, retaining 80 % activity after 30 days. His-HsADH2 showed preference for NADP(H) but interestingly retained 60 % activity towards NADH. The enzyme displayed broad substrate specificity, with maximum activity obtained for 1-propanol. The enzyme also accepted secondary alcohols such as 2-butanol and even 1-phenylethanol. In the reductive reaction, working conditions for His-HsADH2 were optimised for acetaldehyde and found to be 4 M KCl and pH 6.0. His-HsADH2 displayed intrinsic organic solvent tolerance, which is highly relevant for biotechnological applications.

Increase of salt dependence of halophilic nucleoside diphosphate kinase caused by a single amino acid substitution.

Nucleoside diphosphate kinase (HsNDK) from an extremely halophilic archaea, Halobacterium salinarum, is composed of a homo hexamer, assembled as a trimer of basic dimeric units. It requires >2 M NaCl for refolding, although it does not require NaCl for stability or enzymatic activity below 30 °C. A HisN111L mutant with an N-terminal extension sequence containing hexa-His tag, in which Asn111 was replaced with Leu, was designed to be less stable between basic dimeric units. This mutant can lose between 6 and 12 hydrogen bonds between basic dimeric units in the hexamer structure. The HisN111L mutant had enhanced salt requirements for enzymatic activity and refolding even though the secondary structure of the HisN111L mutant was confirmed to be similar to the control, HisNDK, in low and high salt solutions using circular dichroism. We reported previously that G114R and D148C mutants, which had enhanced interactions between basic dimeric units, showed facilitated refolding and stabilization in low salt solution. The results of this study help to elucidate the process for engineering industrial enzymes by controlling subunit-subunit interactions through mutations.

The orphan protein bis-γ-glutamylcystine reductase joins the pyridine nucleotide disulfide reductase family.

Facile DNA sequencing became possible decades after many enzymes had been purified and characterized. Consequently, there are still "orphan" enyzmes for which activities are known but for which encoding genes have not been identified. Identification of the genes encoding orphan enzymes is important because it allows correct annotation of genes of unknown function or with misassigned function. Bis-γ-glutamylcystine reductase (GCR) is an orphan protein that was purified in 1988. This enzyme catalyzes the reduction of bis-γ-glutamylcystine. γ-Glutamylcysteine is the major low-molecular weight thiol in halobacteria. We purified GCR from Halobacterium sp. NRC-1 and identified the sequence of 23 tryptic peptides by nano-liquid chromatography electrospray ionization tandem mass spectrometry. These peptides cover 62% of the protein predicted to be encoded by a gene in Halobacterium sp. NRC-1 that is annotated as mercuric reductase. GCR and mercuric reductase activities were assayed using enzyme that was expressed in Escherichia coli and refolded from inclusion bodies. The enzyme had robust GCR activity but no mercuric reductase activity. The genomes of most, but not all, halobacteria for which whole genome sequences are available have close homologues of GCR, suggesting that there is more to be learned about the low-molecular weight thiols used in halobacteria.

Specific volume and compressibility of bilayer lipid membranes with incorporated Na,K-ATPase.

Ultrasound velocimetry and densitometry methods were used to study the interactions of the Na,K-ATPase with the lipid bilayer in large unilamellar liposomes composed of dioleoyl phosphatidylcholine (DOPC). The ultrasound velocity increased and the specific volume of the phospholipids decreased with increasing concentrations of protein. These experiments allowed us to determine the reduced specific apparent compressibility of the lipid bilayer, which decreased by approx. 11% with increasing concentrations of the Na,K-ATPase up to an ATPase/DOPC molar ratio = 2 × 10⁻4. Assuming that ATPase induces rigidization of the surrounding lipid molecules one can obtain from the compressibility data that 3.7 to 100 times more lipid molecules are affected by the protein in comparison with annular lipids. However, this is in contradiction with the current theories of the phase transitions in lipid bilayers. It is suggested that another physical mechanisms should be involved for explanation of observed effect.

Catalytic and thermodynamic characterization of protease from Halobacterium sp. SP1(1).

Osmolytes KCl, glycerol, mannitol, trehalose, sucrose, betaine, proline and Na-glutamate at different concentrations (5-30%) were investigated as effective solutes for retaining the activity of Halobacterium sp. SP1(1) protease in the absence of NaCl. Maximum activity was observed in the presence of 30% Na-glutamate. Kinetic and thermodynamic parameters for casein hydrolysis revealed that the protease was equally efficient in the presence of Na-glutamate as in NaCl. The enzyme was active over a broader range of temperature (20-80 degrees C) and was highly stable even at 80 degrees C with Na-glutamate. Thermodynamic parameters (DeltaH*, DeltaS*, G*) for irreversible inactivation of protease at different temperatures (20-80 degrees C) were determined in the presence of Na-glutamate and NaCl. The efficiency of these osmolytes for thermal stability of protease was 30% (1.6 M) Na-glutamate > 4 M ( approximately 25%) NaCl > 2 M (approximately 10%), suggesting that the effect exerted by the osmolyte depends not only on its chemical nature but also on its concentration. Na-glutamate was thus found to play an important role in thermal stabilization of enzyme substituting for NaCl. Moreover, substitution of NaCl by Na-glutamate may increase the applicability of halophilic enzymes in biotechnology and industry, which is otherwise limited to high NaCl concentrations.

Carboxyl ester hydrolases production and growth of a halophilic archaeon, Halobacterium sp. NRC-1.

The capability of Halobacterium sp. NRC-1 to synthesize carboxyl ester hydrolases was investigated, and the effect of physicochemical conditions on the growth rate and production of esterases was evaluated. The haloarchaeon synthesized a carboxyl ester hydrolase, confirming the genomic prediction. This enzymatic activity was intracellularly produced as a growth-associated metabolite. Esterase activity was assayed using different p-nitrophenyl-esters and triacyl-glycerides, which showed a preference for hydrolyzing tributyrin. The archaeal growth rate and esterase production were significantly influenced by the pH and the NaCl concentration. An interaction effect between temperature and NaCl was also seen. The maximal growth rate and esterase production found for Halobacterium sp. NRC-1 were 0.136 h(-1) (at 4.2 M NaCl, pH 6 and 44 degrees C) and 1.64 U/l (at 4.6 M NaCl, pH 6 and 30 degrees C), respectively. Furthermore, the effects of NaCl concentration, pH and temperature on enzyme activity were studied. Two maximal esterase activities were elucidated from the intracellular crude extract when it was incubated at different NaCl concentrations (1 M and 5 M) and at different pHs (6 and 7.5). This is the first report that shows experimentally the synthesis of carboxyl ester hydrolases by Halobacterium sp. NRC-1. This enzyme was found to be extremely halophilic (5 M NaCl) and thermophilic (80 degrees C), making it very interesting for future investigations in non-aqueous biocatalysis.

Organic solvent tolerance of Halobacterium sp. SP1(1) and its extracellular protease.

Halophilic archaea belonging to three different genera- Halobacterium, Haloarcula and Haloferax, were isolated from Kandla salt pans. The isolates had an optimum requirement of 25% NaCl for growth. Increase in organic solvent tolerance of isolates was observed at higher NaCl concentrations. Among the three isolates Halobacterium sp. SP1(1) was found to be more tolerant than Haloarcula sp. SP2(2) and Haloferax sp. SP1(2a). The extracellular protease of Halobacterium sp. SP1(1) showed higher solvent tolerance compared to the organism itself. The enzyme was highly tolerant to toluene, xylene, n-decane, n-dodecane and n-undecane, majority of which are frequently used in paints. These findings may help in understanding the mechanism of organic solvent tolerance in halophilic archaea and their application in antifouling coatings. Also, best to our knowledge the present study is the first report on organic solvent tolerance of haloarchaeal extracellular protease.

Rad50 is not essential for the Mre11-dependent repair of DNA double-strand breaks in Halobacterium sp. strain NRC-1.

The genome of the halophilic archaeon Halobacterium sp. strain NRC-1 encodes homologs of the eukaryotic Mre11 and Rad50 proteins, which are involved in the recognition and end processing of DNA double-strand breaks in the homologous recombination repair pathway. We have analyzed the phenotype of Halobacterium deletion mutants lacking mre11 and/or rad50 after exposure to UV-C radiation, an alkylating agent (N-methyl-N'-nitro-N-nitrosoguanidine), and gamma radiation, none of which resulted in a decrease in survival of the mutant strains compared to that of the background strain. However, a decreased rate of repair of DNA double-strand breaks in strains lacking the mre11 gene was observed using pulsed-field gel electrophoresis. These observations led to the hypothesis that Mre11 is essential for the repair of DNA double-strand breaks in Halobacterium, whereas Rad50 is dispensable. This is the first identification of a Rad50-independent function for the Mre11 protein, and it represents a shift in the Archaea away from the eukaryotic model of homologous recombination repair of DNA double-strand breaks.

The two PAN ATPases from Halobacterium display N-terminal heterogeneity and form labile complexes with the 20S proteasome.

The PAN (proteasome-activating nucleotidase) proteins from archaea represent homologues of the eukaryotic 26S proteasome regulatory ATPases. In vitro the PAN complex has been previously shown to have a stimulatory effect on the peptidase activities of the 20S core. By using gradient ultracentrifugation we found that, in cellular extracts, the two PAN proteins from Halobacterium do not form stable high-molecular-mass complexes. Only PAN B was found to associate transiently with the 20S proteasome, thus suggesting that the two PAN proteins are not functionally redundant. The PAN B-20S proteasome complexes associate in an ATP-dependent manner and are stabilized upon nucleotide binding. The two PAN proteins were immunodetected in cellular extracts as N-terminal-truncated polypeptides. RNA-mapping experiments and sequence analysis indicated that this process involved transcript heterogeneities and dual translational initiation mechanisms. Taken together, our results suggest that PAN N-terminal modifications and their intracellular dynamics of assembly/association may constitute important determinants of proteolysis regulation.

Functional analysis of type 1 isopentenyl diphosphate isomerase from Halobacterium sp. NRC-1.

Here we report the characterization of the type-1 isopentenyl diphosphate isomerase derived from Halobacterium sp. NRC-1. The expressed purified enzyme showed maximum isomerase activity in the presence of 1 M NaCl at 37 degrees C at pH 6.0. This type-1 enzyme appears to be the first for which the Co2+ ion is required for activity.

Haloadaptation: insights from comparative modeling studies of halophilic archaeal DHFRs.

Proteins of halophilic archaea function in high-salt concentrations that inactivate or precipitate homologous proteins from non-halophilic species. Haloadaptation and the mechanism behind the phenomenon are not yet fully understood. In order to obtain useful information, homology modeling studies of dihydrofolate reductases (DHFRs) from halophilic archaea were performed that led to the construction of structural models. These models were subjected to energy minimization, structural evaluation and analysis. Complementary approaches concerning calculations of the amino acid composition and visual inspection of the surfaces and cores of the models, as well as calculations of electrostatic surface potentials, in comparison to non-halophilic DHFRs were also performed. The results provide evidence that sheds some light on the phenomenon of haloadaptation: DHFRs from halophilic archaea may maintain their fold, in high-salt concentrations, by sharing highly negatively charged surfaces and weak hydrophobic cores.

Acquisition of an insertion peptide for efficient aminoacylation by a halophile tRNA synthetase.

Enzymes of halophilic organisms contain unusual peptide motifs that are absent from their mesophilic counterparts. The functions of these halophile-specific peptides are largely unknown. Here we have identified an unusual peptide that is unique to several halophile archaeal cysteinyl-tRNA synthetases (CysRS), which catalyze attachment of cysteine to tRNA(Cys) to generate the essential cysteinyl-tRNA(Cys) required for protein synthesis. This peptide is located near the active site in the catalytic domain and is highly enriched with acidic residues. In the CysRS of the extreme halophile Halobacterium species NRC-1, deletion of the peptide reduces the catalytic efficiency of aminoacylation by a factor of 100 that largely results from a defect in kcat, rather than the Km for tRNA(Cys). In contrast, maintaining the peptide length but substituting acidic residues in the peptide with neutral or basic residues has no major deleterious effect, suggesting that the acidity of the peptide is not important for the kcat of tRNA aminoacylation. Analysis of general protein structure under physiological high salt concentrations, by circular dichroism and by fluorescence titration of tRNA binding, indicates little change due to deletion of the peptide. However, the presence of the peptide confers tolerance to lower salt levels, and fluorescence analysis in 30% sucrose reveals instability of the enzyme without the peptide. We suggest that the stability associated with the peptide can be used to promote proper enzyme conformation transitions in various stages of tRNA aminoacylation that are associated with catalysis. The acquisition of the peptide by the halophilic CysRS suggests an enzyme adaptation to high salinity.

Crystallization of halorhodopsin from Halobacterium sp. shark.

The chloride-ion-pumping channel, halorhodopsin from Halobacterium sp. shark was detergent-solubilized and 3-D crystallized. Proteins were solubilized using the nonionic detergent n-octyl-beta-D-glucoside and were crystallized as thin-plate crystals with polyethylene glycol 4000 as a precipitant. The crystals belong to the space group P4(1)2(1)2 with unit-cell dimensions a=b=74.5 A and c=138.6 A. The diffraction pattern was slightly anisotropic. The best ordered crystal diffracted up to 3.3 A resolution along c axis with synchrotron radiation.

Systems level insights into the stress response to UV radiation in the halophilic archaeon Halobacterium NRC-1.

We report a remarkably high UV-radiation resistance in the extremely halophilic archaeon Halobacterium NRC-1 withstanding up to 110 J/m2 with no loss of viability. Gene knockout analysis in two putative photolyase-like genes (phr1 and phr2) implicated only phr2 in photoreactivation. The UV-response was further characterized by analyzing simultaneously, along with gene function and protein interactions inferred through comparative genomics approaches, mRNA changes for all 2400 genes during light and dark repair. In addition to photoreactivation, three other putative repair mechanisms were identified including d(CTAG) methylation-directed mismatch repair, four oxidative damage repair enzymes, and two proteases for eliminating damaged proteins. Moreover, a UV-induced down-regulation of many important metabolic functions was observed during light repair and seems to be a phenomenon shared by all three domains of life. The systems analysis has facilitated the assignment of putative functions to 26 of 33 key proteins in the UV response through sequence-based methods and/or similarities of their predicted three-dimensional structures to known structures in the PDB. Finally, the systems analysis has raised, through the integration of experimentally determined and computationally inferred data, many experimentally testable hypotheses that describe the metabolic and regulatory networks of Halobacterium NRC-1.

Identification of the first archaeal Type 1 RNase H gene from Halobacterium sp. NRC-1: archaeal RNase HI can cleave an RNA-DNA junction.

All the archaeal genomes sequenced to date contain a single Type 2 RNase H gene. We found that the genome of a halophilic archaeon, Halobacterium sp. NRC-1, contains an open reading frame with similarity to Type 1 RNase H. The protein encoded by the Vng0255c gene, possessed amino acid sequence identities of 33% with Escherichia coli RNase HI and 34% with a Bacillus subtilis RNase HI homologue. The B. subtilis RNase HI homologue, however, lacks amino acid sequences corresponding to a basic protrusion region of the E. coli RNase HI, and the Vng0255c has the similar deletion. As this deletion apparently conferred a complete loss of RNase H activity on the B. subtilis RNase HI homologue protein, the Vng0255c product was expected to exhibit no RNase H activity. However, the purified recombinant Vng0255c protein specifically cleaved an RNA strand of the RNA/DNA hybrid in vitro, and when the Vng0255c gene was expressed in an E. coli strain MIC2067 it could suppress the temperature-sensitive growth defect associated with the loss of RNase H enzymes of this strain. These results in vitro and in vivo strongly indicate that the Halobacterium Vng0255c is the first archaeal Type 1 RNase H. This enzyme, unlike other Type 1 RNases H, was able to cleave an Okazaki fragment-like substrate at the junction between the 3'-side of ribonucleotide and 5'-side of deoxyribonucleotide. It is likely that the archaeal Type 1 RNase H plays a role in the removal of the last ribonucleotide of the RNA primer from the Okazaki fragment during DNA replication.

Adaptation of an orthogonal archaeal leucyl-tRNA and synthetase pair for four-base, amber, and opal suppression.

Recently, it has been shown that an amber suppressor tRNA/aminoacyl-tRNA synthetase pair derived from the tyrosyl-tRNA synthetase of Methanococcus jannaschii can be used to genetically encode unnatural amino acids in response to the amber nonsense codon, TAG. However, we have been unable to modify this pair to decode either the opal nonsense codon, TGA, or the four-base codon, AGGA, limiting us to a 21 amino acid code. To overcome this limitation, we have adapted a leucyl-tRNA synthetase from Methanobacterium thermoautotrophicum and leucyl tRNA derived from Halobacterium sp. NRC-1 as an orthogonal tRNA-synthetase pair in Escherichia coli to decode amber (TAG), opal (TGA), and four-base (AGGA) codons. To improve the efficiency and selectivity of the suppressor tRNA, extensive mutagenesis was performed on the anticodon loop and acceptor stem. The two most significant criteria required for an efficient amber orthogonal suppressor tRNA are a CU(X)XXXAA anticodon loop and the lack of noncanonical or mismatched base pairs in the stem regions. These changes afford only weak suppression of TGA and AGGA. However, this information together with an analysis of sequence similarity of multiple native archaeal tRNA sequences led to efficient, orthogonal suppressors of opal codons and the four-base codon, AGGA. Ultimately, it should be possible to use these additional orthogonal pairs to genetically incorporate multiple unnatural amino acids into proteins.

The cobY gene of the archaeon Halobacterium sp. strain NRC-1 is required for de novo cobamide synthesis.

Genetic and nutritional analyses of mutants of the extremely halophilic archaeon Halobacterium sp. strain NRC-1 showed that open reading frame (ORF) Vng1581C encodes a protein with nucleoside triphosphate:adenosylcobinamide-phosphate nucleotidyltransferase enzyme activity. This activity was previously associated with the cobY gene of the methanogenic archaeon Methanobacterium thermoautotrophicum strain DeltaH, but no evidence was obtained to demonstrate the direct involvement of this protein in cobamide biosynthesis in archaea. Computer analysis of the Halobacterium sp. strain NRC-1 ORF Vng1581C gene and the cobY gene of M. thermoautotrophicum strain DeltaH showed the primary amino acid sequence of the proteins encoded by these two genes to be 35% identical and 48% similar. A strain of Halobacterium sp. strain NRC-1 carrying a null allele of the cobY gene was auxotrophic for cobinamide-GDP, a known intermediate of the late steps of cobamide biosynthesis. The auxotrophic requirement for cobinamide-GDP was corrected when a wild-type allele of cobY was introduced into the mutant strain, demonstrating that the lack of cobY function was solely responsible for the observed block in cobamide biosynthesis in this archaeon. The data also show that Halobacterium sp. strain NRC-1 possesses a high-affinity transport system for corrinoids and that this archaeon can synthesize cobamides de novo under aerobic growth conditions. To the best of our knowledge this is the first genetic and nutritional analysis of cobalamin biosynthetic mutants in archaea.