Hyperthermophilic Archaeon Sulfolobus Tokodaii Research Articles

Crystal structure of thermostable acetaldehyde dehydrogenase from the hyperthermophilic archaeon Sulfolobus tokodaii.

Aldehyde dehydrogenase (ALDH) is widely distributed in nature and its characteristics have been examined. ALDH plays an important role in aldehyde detoxification. Sources of aldehydes include incomplete combustion and emissions from paints, linoleum and varnishes in the living environment. Acetaldehyde is also considered to be carcinogenic and toxic. Thermostable ALDH from the hyperthermophilic archaeon Sulfolobus tokodaii exhibits high activity towards acetaldehyde and has potential applications as a biosensor for acetaldehyde. Thermostable ALDH displays a unique and wide adaptability. Therefore, its crystal structure can provide new insights into the catalytic mechanism and potential applications of ALDHs. However, a crystal structure of a thermostable ALDH exhibiting high activity towards acetaldehyde has not been reported to date. In this study, crystals of recombinant thermostable ALDH from S. tokodaii were prepared and the crystal structure of its holo form was determined. A crystal of the enzyme was prepared and its structure in complex with NADP was determined at a resolution of 2.2 Å. This structural analysis may facilitate further studies on catalytic mechanisms and applications.

Activity-stability trade-off in random mutant proteins

In our previous study, we investigated the relationship between protein evolution and stability through the random mutational drift of an esterase from hyperthermophilic archaeon Sulfolobus tokodaii. The results revealed that evolvability, which is the appearance frequency of variants with higher activity than the parent protein, correlates with parental stability. This suggests that protein evolution that does not take stability into account does not make sense. Here, we used those data to further evaluate the relationship between activity and stability in random mutations, revealing that the maximum increase in activity due to mutation conflicts with parental stability. That is, many activated variants are produced when parental stability is high, whereas lower stability offers a few excellent variants with much higher activity. Moreover, we used the random mutant library to compute a novel criterion, robustizability (stabilizability), which is the appearance frequency of variants with a higher stability than the parent protein. Robustizability correlates positively with parental activity and negatively with parental stability. The results indicated that the principle of activity-stability trade-off dominates, in even random mutations. We propose its application in protein engineering via directed evolution by stability selection.

Biochemical characterization of the nuclease StoNurA from the hyperthermophilic archaeon Sulfolobus tokodaii.

The DNA nuclease gene ST2109 has been cloned from the hyperthermophilic archaeon Sulfolobus tokodaii and expressed in Escherichia coli. The recombinant protein StoNurA has been purified to homogeneity by affinity chromatography and gel filtration chromatography. Biochemical analyses demonstrated that StoNurA exhibited DNA binding and 5'-3' exonuclease activities towards ssDNA and dsDNA. The temperature and pH optima of StoNurA were determined to be 65 °C and 8.0, respectively. The activity of StoNurA was found to be dependent of Mn2+, and its half-life of heat inactivation at 100 °C was 5 min. Gel filtration chromatography revealed that StoNurA could form dimers in solution. Pull-down assays also showed that StoNurA physically interacted with a DNA helicase (StoHerA). Our data suggest that NurA may play a key functional role in the processing of DNA recombinational repair.

Protein Evolution is Potentially Governed by Protein Stability: Directed Evolution of an Esterase from the Hyperthermophilic Archaeon Sulfolobus tokodaii.

The study of evolution is important to understand biological phenomena. During evolutionary processes, genetic changes confer amino acid substitutions in proteins, resulting in new or improved functions. Unfortunately, most mutations destabilize proteins. Thus, protein stability is a significant factor in evolution; however, its role remains unclear. Here, we simply and directly explored the association between protein activity and stability in random mutant libraries to elucidate the role of protein stability in evolutionary processes. In the first random mutation of an esterase from Sulfolobus tokodaii, approximately 20% of the variants displayed higher activity than wild-type protein (i.e., 20% evolvability). During evolutionary processes, the evolvability depended on the stability of template proteins, indicating that protein evolution is potentially governed by protein stability. Furthermore, decreased activity could be recovered during evolution by maintaining the stability of variants. The results suggest that protein sequence space for its evolution is able to expand during nearly neutral evolution where mutations are slightly deleterious for activity but rarely fatal for stability. Molecular evolution is a crucial phenomenon that has continued since the birth of life on earth, and mechanism underlying it is simple; therefore, this could be demonstrated by our simple experiments. These findings also can be applied to protein engineering.

Inhibition of homoserine dehydrogenase by formation of a cysteine-NAD covalent complex

Homoserine dehydrogenase (EC 1.1.1.3, HSD) is an important regulatory enzyme in the aspartate pathway, which mediates synthesis of methionine, threonine and isoleucine from aspartate. Here, HSD from the hyperthermophilic archaeon Sulfolobus tokodaii (StHSD) was found to be inhibited by cysteine, which acted as a competitive inhibitor of homoserine with a Ki of 11 μM and uncompetitive an inhibitor of NAD and NADP with Ki’s of 0.55 and 1.2 mM, respectively. Initial velocity and product (NADH) inhibition analyses of homoserine oxidation indicated that StHSD first binds NAD and then homoserine through a sequentially ordered mechanism. This suggests that feedback inhibition of StHSD by cysteine occurs through the formation of an enzyme-NAD-cysteine complex. Structural analysis of StHSD complexed with cysteine and NAD revealed that cysteine situates within the homoserine binding site. The distance between the sulfur atom of cysteine and the C4 atom of the nicotinamide ring was approximately 1.9 Å, close enough to form a covalent bond. The UV absorption-difference spectrum of StHSD with and without cysteine in the presence of NAD, exhibited a peak at 325 nm, which also suggests formation of a covalent bond between cysteine and the nicotinamide ring.

Structural insight into activation of homoserine dehydrogenase from the archaeon Sulfolobus tokodaii via reduction

Homoserine dehydrogenase (HSD; 305 amino acid residues) catalyzes an NAD(P)-dependent reversible reaction between l-homoserine and aspartate 4-semialdehyde and is involved in the aspartate pathway. HSD from the hyperthermophilic archaeon Sulfolobus tokodaii was markedly activated (2.5-fold) by the addition of 0.8mM dithiothreitol. The crystal structure of the homodimer indicated that the activation was caused by cleavage of the disulfide bond formed between two cysteine residues (C303) in the C-terminal regions of the two subunits.

Molecular characterization of a thermostable aldehyde dehydrogenase (ALDH) from the hyperthermophilic archaeon Sulfolobus tokodaii strain 7

Aldehyde dehydrogenase (ALDH) is a widely distributed enzyme in nature. Although many ALDHs have been reported until now, the detailed enzymatic properties of ALDH from Archaea remain elusive. Herein, we describe the characterization of an ALDH from the hyperthermophilic archaeon Sulfolobus tokodaii. The enzyme (stALDH) could utilize various aldehydes as substrates, and maximal activity was found with acetaldehyde and the coenzyme NAD. The optimal temperature and pH were 80°C and 8, respectively, and high thermostability was found with the half-life at 90°C to be 4h. The enzyme was considerably resistant to nitroglycerin (GTN) inhibition, which could be restored by reducing agent DTT or (±)-α-lipoic acid. Coenzyme NAD or NADP could regulate the enzymatic thermostability, as well as the esterase activity. Molecular modeling suggested that the enzyme harbored similar structural arrangement with its eukaryotic and bacterial counterparts. Sequence alignment showed the conserved catalytic residues E240 and C274 and cofactor interactive sites N142, K165, I168 and E370, the function of which were verified by site-directed mutagenesis analysis. This is the most thermostable ALDH reported until now and the unique property of this enzyme is potentially beneficial in the fields of biotechnology and biomedicine.

Dissection of the functional domains of an archaeal Holliday junction helicase

Helicases and nucleases form complexes that play very important roles in DNA repair pathways some of which interact with each other at Holliday junctions. In this study, we present in vitro and in vivo analysis of Hjm and its interaction with Hjc in Sulfolobus. In vitro studies employed Hjm from the hyperthermophilic archaeon Sulfolobus tokodaii (StoHjm) and its truncated derivatives, and characterization of the StoHjm proteins revealed that the N-terminal module (residues 1–431) alone was capable of ATP hydrolysis and DNA binding, while the C-terminal one (residues 415–704) was responsible for regulating the helicase activity. The region involved in StoHjm–StoHjc (Hjc from S. tokodaii) interaction was identified as part of domain II, domain III (Winged Helix motif), and domain IV (residues 366–645) for StoHjm. We present evidence supporting that StoHjc regulates the helicase activity of StoHjm by inducing conformation change of the enzyme. Furthermore, StoHjm is able to prevent the formation of Hjc/HJ high complex, suggesting a regulation mechanism of Hjm to the activity of Hjc. We show that Hjm is essential for cell viability using recently developed genetic system and mutant propagation assay, suggesting that Hjm/Hjc mediated resolution of stalled replication forks is of crucial importance in archaea. A tentative pathway with which Hjm/Hjc interaction could have occurred at stalled replication forks is discussed.

Characterization of the specific interaction between archaeal FHA domain-containing protein and the promoter of a flagellar-like gene-cluster and its regulation by phosphorylation

The mechanism and target genes of regulation by Forkhead (FHA) domain-containing transcription factors have not yet been documented in Archaea. In this study, using a bacterial one-hybrid technique, we successfully screened and identified for the first time a target gene regulated by ST0829, an FHA domain-containing potential transcriptional factor in the hyperthermophilic archaeon Sulfolobus tokodaii. We show that ST0829 could specifically bind to the promoter region of ST2519p, the archaeal flagellar protein-encoding operon (including FlaG, FlaF, FlaH, FlaI, and FlaJ) by using both in vitro electrophoretic mobility shift assay and surface plasmon resonance experiments, and in vivo chromatin immunoprecipitation assays. Furthermore, phosphorylation of the FHA domain-containing protein was found to negatively regulate its specific DNA-binding activity. The interaction between ST0829 and ST2519p could be inhibited by wild-type Ser/Thr protein kinase ST1565, but was not significantly affected by its mutant variant ST1565-K166A that lacks kinase activity. These findings not only increase our knowledge about the function of an archaeal FHA domain-containing regulator but also offer important insights for further understanding the signaling mechanism of environmental adaptation in archaea.

Evolution and thermodynamics of the slow unfolding of hyperstable monomeric proteins

BackgroundThe unfolding speed of some hyperthermophilic proteins is dramatically lower than that of their mesostable homologs. Ribonuclease HII from the hyperthermophilic archaeon Thermococcus kodakaraensis (Tk-RNase HII) is stabilized by its remarkably slow unfolding rate, whereas RNase HI from the thermophilic bacterium Thermus thermophilus (Tt-RNase HI) unfolds rapidly, comparable with to that of RNase HI from Escherichia coli (Ec-RNase HI).ResultsTo clarify whether the difference in the unfolding rate is due to differences in the types of RNase H or differences in proteins from archaea and bacteria, we examined the equilibrium stability and unfolding reaction of RNases HII from the hyperthermophilic bacteria Thermotoga maritima (Tm-RNase HII) and Aquifex aeolicus (Aa-RNase HII) and RNase HI from the hyperthermophilic archaeon Sulfolobus tokodaii (Sto-RNase HI). These proteins from hyperthermophiles are more stable than Ec-RNase HI over all the temperature ranges examined. The observed unfolding speeds of all hyperstable proteins at the different denaturant concentrations studied are much lower than those of Ec-RNase HI, which is in accordance with the familiar slow unfolding of hyperstable proteins. However, the unfolding rate constants of these RNases H in water are dispersed, and the unfolding rate constant of thermophilic archaeal proteins is lower than that of thermophilic bacterial proteins.ConclusionsThese results suggest that the nature of slow unfolding of thermophilic proteins is determined by the evolutionary history of the organisms involved. The unfolding rate constants in water are related to the amount of buried hydrophobic residues in the tertiary structure.

The first crystal structure of an archaeal metallo‐β‐lactamase superfamily protein; ST1585 from Sulfolobus tokodaii

The first crystal structure of an archaeal metallo‐β‐lactamase superfamily protein; ST1585 from <i>Sulfolobus tokodaii</i>

Spatial subunit distribution and in vitro functions of the novel trimeric PCNA complex from Sulfolobus tokodaii

The relationships among three PCNA (proliferating cell nuclear antigen) subunits in the hyperthermophilic archaeon Sulfolobus tokodaii (StoPCNAs) were analyzed and the effects of two PCNA complexes on the activities of the DNA helicase Hjm, DNA Ligase I, and Holliday junction specific endonuclease Hjc were tested. There was no strong self-interaction of each StoPCNA. StoPCNA1 and StoPCNA3 interacted with each other, so did StoPCNA2 and StoPCNA3, but no interaction between StoPCNA1 and StoPCNA2 was observed. Two trimeric complexes (designed StoPCNA123 and StoPCNA323) were formed in vitro and it was determined that StoPCNA323 was composed of one StoPCNA2 and two StoPCNA3 subunits, with StoPCNA2 bridging the two StoPCNA3 subunits. Both complexes inhibited the unwinding activity of Hjm and the ligation activity of DNA Ligase I. In contrast, both stimulated the Holliday junction cleavage activity of Hjc. Our results provide further evidence that in crenarchaea, the PCNAs exhibit diversity in subunit interaction and complex formation.

Archaeal DNA helicase HerA interacts with Mre11 homologue and unwinds blunt-ended double-stranded DNA and recombination intermediates

HerA is a novel family DNA helicases that exist ubiquitously in thermophilic archaea. The genes are linked to homologues of eukaryotic recombination and repair proteins Mre11 and Rad50 in some of the genomes. However, the relationship between HerA and the related proteins is unclear. In this study, a homologue from the hyperthermophilic archaeon Sulfolobus tokodaii (StoHerA) was characterized and physical and functional interactions between StoHerA and StoMre11 (Mre11 from S. tokodaii) were studied. It was found that StoHerA was able to unwind blunt-ended double-stranded DNA (dsDNA), although with lower efficiency. StoHerA was also able to unwind Holliday junction, splayed-arm DNA, as well as 5′- or 3′-overhang with high efficiency. Pull-down and yeast two-hybrid analyses revealed that StoHerA interacted with StoMre11 physically. The helicase activity of StoHerA was stimulated by StoMre11, indicating a functional role of this interaction. In addition, site-directed mutagenesis of StoHerA was performed to analyze functions of conserved residues of StoHerA. Interestingly, mutation of E355 to alanine in Walker B resulted in not only loss of ATPase and DNA helicase activities, but also dsDNA-binding ability, indicating that this residue is involved in the coupling of ATP hydrolysis, dsDNA-binding, and helicase activities.

Structure of l-aspartate oxidase from the hyperthermophilic archaeon Sulfolobus tokodaii

The crystal structure of the highly thermostable l-aspartate oxidase (LAO) from the hyperthermophilic archaeon Sulfolobus tokodaii was determined at a 2.09 Å resolution. The factors contributing to the thermostability of the enzyme were analyzed by comparing its structure to that of Escherichia coli LAO. Like E. coli LAO, the S. tokodaii enzyme consists of three domains: an FAD-binding domain, an α + β capping domain, and a C-terminal three-helix bundle. However, the situation of the linker between the FAD-binding domain and C-terminal three-helix bundle in S. tokodaii LAO is completely different from that in E. coli LAO, where the linker is situated near the FAD-binding domain and has virtually no interaction with the rest of the protein. In S. tokodaii LAO, this linker is situated near the C-terminal three-helix bundle and contains a β-strand that runs parallel to the C-terminal strand. This results in the formation of an additional β-sheet, which appears to reduce the flexibility of the C-terminal region. Furthermore, the displacement of the linker enables formation of a 5-residue ion-pair network between the FAD-binding and C-terminal domains, which strengthens the interdomain interactions. These features might be the main factors contributing to the high thermostability of S. tokodaii LAO.

Molecular and functional characterization of d-3-phosphoglycerate dehydrogenase in the serine biosynthetic pathway of the hyperthermophilic archaeon Sulfolobus tokodaii

A gene (ST1218) encoding a d-3-phosphoglycerate dehydrogenase (PGDH; EC 1.1.1.95) homolog was found in the genome of Sulfolobus tokodaii strain 7 by screening a database of enzymes likely to contribute to l-serine biosynthesis in hyperthermophilic archaea. After expressing the gene in Escherichia coli, the PGDH activity of the recombinant enzyme was assessed. Homogeneous PGDH was obtained using conventional chromatography steps, though during the purification an unexpected decline in enzyme activity was observed if the enzyme was stored in plastic tubes, but not in glass ones. The purified enzyme was a homodimer with a subunit molecular mass of about 35 kDa and was highly thermostable. It preferably acted as an NAD-dependent d-3-phosphoglycerate (3PGA) dehydrogenase. Although NADP had no activity as the electron acceptor, both NADPH and NADH acted as electron donors. Kinetic analyses indicated that the enzyme reaction proceeds via a Theorell-Chance Bi-Bi mechanism. Unlike E. coli PGDH, the S. tokodaii enzyme was not inhibited by l-serine. In addition, both the NAD-dependent 3PGA oxidation and the reverse reaction were enhanced by phosphate and sulfate ions, while NADPH-dependent 3-phosphohydroxypyruvate (PHP) reduction was inhibited. Thus S. tokodaii PGDH appears to be subject to a novel regulatory mechanism not seen elsewhere. A database analysis showed that ST1218 gene forms a cluster with ST1217 gene, and a functional analysis of the ST1217 product expressed in E. coli revealed that it possesses l-glutamate-PHP aminotransferase activity. Taken together, our findings represent the first example of a phosphorylated serine pathway in a hyperthermophilic archaeon.

Physical and functional interaction between archaeal single-stranded DNA-binding protein and the 5′–3′ nuclease NurA

NurA is a novel 5′–3′ exonuclease that is closely linked to Mre11 and Rad50 homologues in most thermophilic archaea. We report a physical and functional interaction between NurA (StoNurA) and single-stranded DNA-binding protein (StoSSB) from the hyperthermophilic archaeon Sulfolobus tokodaii. StoSSB was identified as a novel StoNurA-interacting protein by pull-down assay using Ni-NTA agarose beads and MALDI-TOF mass spectrometry. The direct interaction between StoNurA and StoSSB was further confirmed by yeast two-hybrid and co-immunoprecipitation analysis. The interaction was supposed to have functional significance because it was found that StoSSB inhibited the 5′–3′ ssDNA and dsDNA exonuclease and ssDNA endonuclease activities of StoNurA. Our results suggest that NurA may function closely together with SSB in DNA transactions in archaea.

Crystal Structure of Type 1 Ribonuclease H from Hyperthermophilic ArchaeonSulfolobus tokodaii: Role of Arginine 118 and C-Terminal Anchoring,

The crystal structure of ribonuclease HI from the hyperthermophilic archaeon Sulfolobus tokodaii (Sto-RNase HI) was determined at 1.6 A resolution. Sto-RNase HI exhibits not only RNase H activity but also double-stranded RNA-dependent ribonuclease (dsRNase) activity. The main-chain fold and steric configurations of the four acidic active-site residues of Sto-RNase HI are very similar to those of other type 1 RNases H. However, Arg118 of Sto-RNase HI is located at the position in which His124 of E. coli RNase HI, His539 of HIV-1 RNase H, and Glu188 of Bacillus halodurans RNase H are located. The mutation of this residue to Ala considerably reduced both the RNase H and dsRNase activities without seriously affecting substrate binding, suggesting that Arg118 is involved in catalytic function. This residue may promote product release by perturbing the coordination of the metal ion A as proposed for Glu188 of B. halodurans RNase H. In addition, the extreme C-terminal region of Sto-RNase HI is anchored to its core region by one disulfide bond and several hydrogen bonds. Differential scanning calorimetry measurements indicated that Sto-RNase HI is a hyperstable protein with a melting temperature of 102 degrees C. The mutations of the cysteine residues forming disulfide bond or elimination of the extreme C-terminal region greatly destabilized the protein, indicating that anchoring of the C-terminal tail is responsible for hyperstabilization of Sto-RNase HI.

Crystal structure of an archaeal homologue of multidrug resistance repressor protein, EmrR, from hyperthermophilic archaea Sulfolobus tokodaii strain 7

MarR family proteins, MarR, MexR, and EmrR, are known as bacterial regulators for a phenotype resistant to multiple antibiotic drugs. Genomic data have indicated the presence of bacterial-type transcriptional regulators, including MarR family proteins in archaea, though the archaeal transcription system is close to that of eukaryote. To elucidate the structural basis of the transcriptional regulation mechanism of archaeal MarR family proteins, the crystal structure of the ST1710 protein, which was identified as an archaeal EmrR homologue, StEmrR, from hyperthermophilic archaeon Sulfolobus tokodaii strain 7 was determined at 1.45-A resolution. The protein was composed of two N- and C-terminal dimerization domains, and the DNA-binding domain consisted of a winged helix motif, as in the case of bacterial MarR family proteins. Despite the relatively low overall structural similarity between StEmrR and bacterial MarR family proteins, the structure of the DNA-binding domain displayed high structural similarity. A comparison with the crystal structures of bacterial MarR family proteins revealed that structural variation was mainly due to the different orientation of the two helices at the N- and C-termini. Our results indicated that the distance between the two DNA-binding domains of MarR family proteins would be changed by the rotation of the two terminal helices to interact with the target DNA.

Crystal Structures of an ATP-dependent Hexokinase with Broad Substrate Specificity from the Hyperthermophilic Archaeon Sulfolobus tokodaii

Hexokinase catalyzes the phosphorylation of glucose to glucose 6-phosphate by using ATP as a phosphoryl donor. Recently, we identified and characterized an ATP-dependent hexokinase (StHK) from the hyperthermophilic archaeon Sulfolobus tokodaii, which can phosphorylate a broad range of sugar substrates, including glucose, mannose, glucosamine, and N-acetylglucosamine. Here we present the crystal structures of StHK in four different forms: (i) apo-form, (ii) binary complex with glucose, (iii) binary complex with ADP, and (iv) quaternary complex with xylose, Mg(2+), and ADP. Forms i and iii are in the open state, and forms ii and iv are in the closed state, indicating that sugar binding induces a large conformational change, whereas ADP binding does not. The four different crystal structures of the same enzyme provide "snapshots" of the conformational changes during the catalytic cycle. StHK exhibits a core fold characteristic of the hexokinase family, but the structures of several loop regions responsible for substrate binding are significantly different from those of other known hexokinase family members. Structural comparison of StHK with human N-acetylglucosamine kinase and other hexokinases provides an explanation for the ability of StHK to phosphorylate both glucose and N-acetylglucosamine. A Mg(2+) ion and coordinating water molecules are well defined in the electron density of the quaternary complex structure. This structure represents the first direct visualization of the binding mode for magnesium to hexokinase and thus allows for a better understanding of the catalytic mechanism proposed for the entire hexokinase family.

Gene expression and characterization of 2-keto-3-deoxygluconate kinase, a key enzyme in the modified Entner–Doudoroff pathway of the aerobic and acidophilic hyperthermophile Sulfolobus tokodaii

2-Keto-3-deoxygluconate kinase (KDGK) catalyzes the ATP-dependent phosphorylation of 2-keto-3-deoxygluconate, a key intermediate in the modified (semi-phosphorylative) Entner–Doudoroff (ED) glucose metabolic pathway. We identified the gene (ORF ID: ST2478) encoding KDGK in the hyperthermophilic archaeon Sulfolobus tokodaii based on the structure of a gene cluster in a genomic database and functionally expressed it in Escherichia coli. The expressed protein was purified from crude extract by heat treatment and two conventional column chromatography steps, and the partial amino acid sequence in the N-terminal region of the purified enzyme (MAKLIT) was identical to that obtained from the gene sequence. The purified enzyme was extremely thermostable and retained full activity after heating at 80 °C for 1 h. The enzyme utilized ATP or GTP, but not ADP or AMP, as a phosphoryl donor and 2-keto-3-deoxy- d-gluconate or 2-keto- d-gluconate as a phosphoryl acceptor. Divalent cations including Mg 2+, Co 2+, Ni 2+, Zn 2+ or Mn 2+ were required for activity, and the apparent Km values for KDG and ATP at 50 °C were 0.027 mM and 0.057 mM, respectively. The presence of KDGK means that the hyperthermophilic archaeon S. tokodaii metabolizes glucose via both modified (semi-phosphorylative) and non-phosphorylative ED pathways.