Crystal structure of full length topoisomerase I from Thermotoga maritima

DNA topoisomerases are a family of enzymes altering the topology of DNA by concerted breakage and rejoining of the phosphodiester backbone of DNA. Bacterial and archeal type IA topoisomerases, including topoisomerase I, topoisomerase III, and reverse gyrase, are crucial in regulation of DNA supercoiling and maintenance of genetic stability. The crystal structure of full length topoisomerase I from Thermotoga maritima was determined at 1.7A resolution and represents an intact and fully active bacterial topoisomerase I. It reveals the torus-like structure of the conserved transesterification core domain comprising domains I-IV and a tightly associated C-terminal zinc ribbon domain (domain V) packing against domain IV of the core domain. The previously established zinc-independence of the functional activity of T.maritima topoisomerase I is further supported by its crystal structure as no zinc ion is bound to domain V. However, the structural integrity is preserved by the formation of two disulfide bridges between the four Zn-binding cysteine residues. A functional role of domain V in DNA binding and recognition is suggested and discussed in the light of the structure and previous biochemical findings. In addition, implications for bacterial topoisomerases I are provided.     

A novel member of the YchN-like fold: solution structure of the hypothetical protein Tm0979 from Thermotoga maritima

We report herein the NMR structure of Tm0979, a structural proteomics target from Thermotoga maritima. The Tm0979 fold consists of four beta/alpha units, which form a central parallel beta-sheet with strand order 1234. The first three helices pack toward one face of the sheet and the fourth helix packs against the other face. The protein forms a dimer by adjacent parallel packing of the fourth helices sandwiched between the two beta-sheets. This fold is very interesting from several points of view. First, it represents the first structure determination for the DsrH family of conserved hypothetical proteins, which are involved in oxidation of intracellular sulfur but have no defined molecular function. Based on structure and sequence analysis, possible functions are discussed. Second, the fold of Tm0979 most closely resembles YchN-like folds; however the proteins that adopt these folds differ in secondary structural elements and quaternary structure. Comparison of these proteins provides insight into possible mechanisms of evolution of quaternary structure through a simple mechanism of hydrophobicity-changing mutations of one or two residues. Third, the Tm0979 fold is found to be similar to flavodoxin-like folds and beta/alpha barrel proteins, and may provide a link between these very abundant folds and putative ancestral half-barrel proteins.     

Cloning and biochemical characterization of Staphylococcus aureus type IA DNA topoisomerase comprised of distinct five domains

DNA topoisomerases play critical roles in regulating DNA topology and are essential enzymes for cell survival. In this study, a gene encoding type IA DNA topoisomerase was cloned from Staphylococcus aureus (S. aureus) sp. strain C-66, and the biochemical properties of recombinant enzyme was characterized. The nucleotide sequence analysis showed that the cloned gene contained an open reading frame (2070 bp) that could encode a polypeptide of 689 amino acids. The cloned gene actually produced 79.1 kDa functional enzyme (named Sau-TopoI) in Escherichia coli (E. coli). Sau-TopoI enzyme purified from E. coli showed ATP-independent and Mg²⁺-dependent manners for relaxing negatively supercoiled DNA. The relaxation activity of Sau-TopoI was inhibited by camptothecin, but not by nalidixic acid and etoposide. Cleavage site mapping showed that the enzyme could preferentially bind to and cleave the sequence GGNN↓CAT (N and ↓ represent any nucleotide and cleavage site, respectively). All these results suggest that the purified enzyme is type IA DNA topoisomerase. In addition, domain mapping analysis showed that the enzyme was composed of conserved four domains (I through IV), together with a variable C-terminal region containing a unique domain V.     

Inhibition of Mycobacterium tuberculosis topoisomerase I by m-AMSA,a eukaryotic type II topoisomerase poison

m-AMSA, an established inhibitor of eukaryotic type II topoisomerases, exerts its cidal effect by binding to the enzyme–DNA complex thus inhibiting the DNA religation step. The molecule and its analogues have been successfully used as chemotherapeutic agents against different forms of cancer. After virtual screening using a homology model of the Mycobacterium tuberculosis topoisomerase I, we identified m-AMSA as a high scoring hit. We demonstrate that m-AMSA can inhibit the DNA relaxation activity of topoisomerase I from M. tuberculosis and Mycobacterium smegmatis. In a whole cell assay, m-AMSA inhibited the growth of both the mycobacteria.     

Unique metal dependency of cytosolic alpha-mannosidase from Thermotoga maritima,a hyperthermophilic bacterium

A putative cytosolic alpha-mannosidase gene from a hyperthermophilic marine bacterium Thermotoga maritima was cloned and expressed in Escherichia coli. The purified recombinant enzyme appeared to be a homodimer of a 110-kDa subunit. The enzyme showed metal-dependent ability to hydrolyze p-nitrophenyl-alpha-D-mannopyranoside. In the absence of a metal, the enzyme was inactive. Cobalt and cadmium supported high activity (60 U/mg at 70 degrees C), while the activity with zinc and chromium was poor. Cobalt (0.8 mol) bound to 1 mol monomer with a K(d) of 70 microM. The optimum pH and temperature were 6.0 and 80 degrees C, respectively. The activity was inhibited by swainsonine, but not by 1-deoxymannojirimycin, which is in agreement with the features of cytosolic alpha-mannosidase.     

Cloning expression and characterization of a thermostable exopolygalacturonase from Thermotoga maritima

A gene encoding for a thermostable exopolygalacturonase (exo-PG) from hyperthermophilic Thermotoga maritima has been cloned into a T7 expression vector and expressed in Escherichia coli. The gene encoded a polypeptide of 454 residues with a molecular mass of 51,304 Da. The recombinant enzyme was purified to homogeneity by heat treatment and nickel affinity chromatography. The thermostable enzyme had maximum of hydrolytic activity for polygalacturonate at 95 degrees C, pH 6.0 and retains 90% of activity after heating at 90 degrees C for 5 h. Study of the catalytic activity of the exopolygalacturonase, investigated by means of 1H NMR spectroscopy revealed an inversion of configuration during hydrolysis of alpha-(1-->4)-galacturonic linkage.     

Structural insight into the low affinity between Thermotoga maritima CheA and CheB compared to their Escherichia coli/Salmonella typhimurium counterparts

CheA-mediated CheB phosphorylation and the subsequent CheB-mediated demethylation of the chemoreceptors are important steps required for the bacterial chemotactic adaptation response. Although Escherichia coli CheB has been reported to interact with CheA competitively against CheY, we have observed that Thermotoga maritima CheB has no detectable CheA-binding. By determining the CheY-like domain crystal structure of T. maritima CheB, and comparing against the T. maritima CheY and Salmonella typhimurium CheB structures, we propose that the two consecutive glutamates in the β4/α4 loop of T. maritima CheB that is absent in T. maritima CheY and in E. coli/S. typhimurium CheB may be one factor contributing to the low CheA affinity.     

Thermotoga maritima IscU. Structural characterization and dynamics of a new class of metallochaperone

Members of the IscU family of proteins are among the most conserved of all protein groups, extending across all three kingdoms of life. IscU serves as a scaffold for the assembly of intermediate iron-sulfur cluster centers and further mediates delivery to apo protein targets. Several proteins that mediate delivery of single metal ions to apo targets (termed metallochaperones) have recently been characterized structurally. Each displays a ferredoxin-like betaalphabetabetaalphabeta motif as a structural core. Assembly and delivery of a polynuclear iron-sulfur cluster is, however, a more complex pathway and presumably would demand a distinctive protein mediator. Here, we demonstrate Thermotoga maritima IscU (Tm IscU) to display unique structural and motional characteristics that distinguish it from other members of this class of proteins. In particular, IscU adopts a mobile, physiologically relevant, molten globule-like state that is vastly different from the previously identified ferredoxin-like fold that has thus far been characterized for other metallochaperones. The secondary structural content of Tm IscU is consistent with previous circular dichroism measurements on apo and holo protein, consisting of six alpha-helices and three beta-strands, the latter forming an anti-parallel beta-sheet. Extensive dynamics studies are consistent with a protein that has reasonably well defined secondary structural elements, but with a tertiary structure that is fluxional among widely different conformational arrangements. Analogous conformational flexibility does not exist in other structurally characterized metallochaperones; however, such a dynamic molecule may account for the lack of long-range NOEs, and allow both for the flexibility that is necessary for the multiple roles of Fe-S cluster assembly, and recognition and delivery of that cluster to a target protein. Additionally, the fluxionality of IscU is unique in that the protein appears to be more compact (based on 1H/2H exchange, R1, R2, and NOE data) but yet more fluid (lack of long-range NOEs) than typical molten globule proteins.     

NMR structure of the conserved hypothetical protein TM0487 from Thermotoga maritima: implications for 216 homologous DUF59 proteins

The NMR structure of the conserved hypothetical protein TM0487 from Thermotoga maritima represents an alpha/beta-topology formed by the regular secondary structures alpha1-beta1-beta2-alpha2-beta3-beta4-alpha3- beta5-3(10)-alpha4, with a small anti-parallel beta-sheet of beta-strands 1 and 2, and a mixed parallel/anti-parallel beta-sheet of beta-strands 3-5. Similar folds have previously been observed in other proteins, with amino acid sequence identity as low as 3% and a variety of different functions. There are also 216 sequence homologs of TM0487, which all have the signature sequence of domains of unknown function 59 (DUF59), for which no three-dimensional structures have as yet been reported. The TM0487 structure thus presents a platform for homology modeling of this large group of DUF59 proteins. Conserved among most of the DUF59s are 13 hydrophobic residues, which are clustered in the core of TM0487. A putative active site of TM0487 consisting of residues D20, E22, L23, T51, T52, and C55 is conserved in 98 of the 216 DUF59 sequences. Asp20 is buried within the proposed active site without any compensating positive charge, which suggests that its pK(a) value may be perturbed. Furthermore, the DUF59 family includes ORFs that are part of a conserved chromosomal group of proteins predicted to be involved in Fe-S cluster metabolism.     

A universal cloning vector using vaccinia topoisomerase I

Vaccinia DNA topoisomerase I (TOPO) charged vectors with a sticky T are routinely used to clone polymerase chain reaction (PCR) products with an extra A at their 3′ end (TOPO TA Cloning from Invitrogen). TOPO charged blunt vectors are used to clone blunt end PCR products (TOPO Blunt Cloning). Here, we demonstrate that both TOPO TA vectors and TOPO Blunt vectors can be used to clone PCR products with either a blunt end or an extra A at the 3′ end. We further demonstrate that these vectors can be used to clone sticky end DNA generated with restriction enzymes. In summary, these TOPO vectors can be used as universal cloning vectors.     

Binding of TmHU to single dsDNA as observed by optical tweezers

Optical tweezers are employed to study the action of the histone-like protein from Thermotoga maritima (TmHU) on DNA at a single molecule level. Binding and disruption of TmHU to and from DNA are found to take place in discrete steps of 4-5 nm length and a net binding enthalpy of about 16kBT. This is in reasonable agreement with a microscopic model that estimates the extension of the binding sites of the protein and evaluates the energetics mainly for bending of the DNA in the course of interaction.     

Enzymatic synthesis of c-di-GMP using a thermophilic diguanylate cyclase

The cyclic dinucleotide c-di-GMP is a widespread bacterial messenger molecule with potential application as a therapeutic agent for treating bacterial infection. Current enzymatic synthesis of c-di-GMP using mesophilic diguanylate cyclase (DGC) proteins suffers from low production yield due to protein instability and strong product inhibition. Here we report the overexpression and characterization of a stand-alone thermophilic diguanylate cyclase domain (tDGC) protein with enhanced thermostability. The product inhibition that severely limited production yield was significantly alleviated by mutation of a conserved residue in the putative regulatory I-site. With the mutant tDGC, we demonstrated that hundreds of milligrams of c-di-GMP can be readily prepared by using the optimized procedures for enzymatic reaction and product purification. The thermophilic enzyme will be a valuable tool for other research laboratories for c-di-GMP synthesis as well as the preparation of c-di-GMP derivatives.     

A simple assay for determining activities of phosphopentomutase from a hyperthermophilic bacterium Thermotoga maritima

Phosphopentomutase (PPM) catalyzes the interconversion of α-d-(deoxy)-ribose 1-phosphate and α-d-(deoxy)-ribose 5-phosphate. We developed a coupled or uncoupled enzymatic assay with an enzyme nucleoside phosphorylase for determining PPM activities on d-ribose 5-phosphate at a broad temperature range from 30 to 90 °C. This assay not only is simple and highly sensitive but also does not require any costly special instrument. Via this technology, an open reading frame TM0167 from a thermophilic bacterium Thermotoga maritima putatively encoding PPM was cloned. The recombinant PPM was overexpressed in Escherichia coli Rosetta. This enzyme has the highest activity at 90 °C. MnCl₂ (0.1 mM) and 50 μM α-d-glucose 1,6-bisphosphate are cofactors. The kinetic parameters of K_m and k_cat are 1.2 mM and 185 s⁻¹ at 90 °C_, respectively. The enzyme has a half-life time of up to 156 min at 90 °C. This enzyme is the most active and thermostable PPM reported to date.     

A novel member of the split betaalphabeta fold: Solution structure of the hypothetical protein YML108W from Saccharomyces cerevisiae

As part of the Northeast Structural Genomics Consortium pilot project focused on small eukaryotic proteins and protein domains, we have determined the NMR structure of the protein encoded by ORF YML108W from Saccharomyces cerevisiae. YML108W belongs to one of the numerous structural proteomics targets whose biological function is unknown. Moreover, this protein does not have sequence similarity to any other protein. The NMR structure of YML108W consists of a four-stranded beta-sheet with strand order 2143 and two alpha-helices, with an overall topology of betabetaalphabetabetaalpha. Strand beta1 runs parallel to beta4, and beta2:beta1 and beta4:beta3 pairs are arranged in an antiparallel fashion. Although this fold belongs to the split betaalphabeta family, it appears to be unique among this family; it is a novel arrangement of secondary structure, thereby expanding the universe of protein folds.     

Enhancement of transglycosylation activity by construction of chimeras between mesophilic and thermophilic beta-glucosidase

The family 3 beta-glucosidase from Thermotoga maritima is a highly thermostable enzyme (85 degrees C) that displays transglycosylation activity. In contrast, the beta-glucosidase from Cellvibrio gilvus is mesophilic (35 degrees C) and displays no such transglycosylation activity. Both enzymes consist of two domains, an N-terminal and a C-terminal domain, and the amino acid identities between the two enzymes in these domains are 32.4 and 36.4%, respectively. In an attempt to identify the molecular basis underpinning the display of transglycosylation activity and the requirements for thermal stability, eight chimeric genes were constructed by shuffling the two parental beta-glucosidase genes at four selected borders, two in the N-terminal domain and two in the C-terminal domain. Of the eight chimeric genes constructed, only two chimeric enzymes (Tm578/606Cg and Tm638/666Cg) gave catalytically active forms and these were the ones shuffled in the C-terminal domain. For these active chimeric enzymes, 80% (Tm578/606Cg) and 88% (Tm638/666Cg) of their amino acid sequences originated from T. maritima. With regard to their thermal profiles, the two active chimeric enzymes, Tm578/606Cg and Tm638/666Cg, displayed profiles intermediate to those of the two parental enzymes as they were optimally active at 65 and 70 degrees C, respectively. These two chimeric enzymes were optimally active at pH 4.1 and 3.9, which is closer to that observed for the T. maritima enzyme (pH 3.2-3.5) than that for the C. gilvus enzyme (pH 6.2-6.5). Kinetic parameters for the chimeric enzymes were investigated with five different substrates including pNP-beta-D-glucopyranoside. The kinetic parameters obtained for the chimeric enzymes were closer to those of the T. maritima enzyme than to those of the C. gilvus enzyme. Transglycosylation activity was observed for both chimeric enzymes and the activity of the Tm578/606Cg chimera was at a level twice that observed with the T. maritima enzyme. This study is an effective demonstration of the usefulness of chimeric enzymes in altering the characteristics of an enzyme.     

Exopolygalacturonate lyase from Thermotoga maritima: cloning,characterization and organic synthesis application

A new exopolygalacturonate lyase (Pel) gene of the hyperthermophilic bacterium Thermotoga maritima was cloned and overexpressed in Escherichia coli cells. A 42 kDa monomeric Pel was shown to undergo N-terminal processing by cleavage at a putative site between alanine and serine residues. The enzyme catalyzes selectively a beta-4,5 elimination at the third galacturonic unit from the reducing end of polygalacturonic acid by producing (4-deoxy-alpha-L-threo-hex-4-enopyranosyluronic acid)-(1-->4)-(alpha-D-galactopyranosyluronic acid)-(1-->4)-alpha-D-galactopyranuronic acid (3) with a 60% yield. The optimum activity of the enzyme was detected at pH 9.5 and T> or=95 degrees C. The highly thermostable enzyme constitutes a useful catalyst for a simplified synthesis of 4,5-unsaturated trigalacturonic acid 3, a trisaccharide which is extremely difficult to obtain via chemical synthesis.     

Crystal structure of Thermotoga maritima 4-alpha-glucanotransferase and its acarbose complex: implications for substrate specificity and catalysis

4-alpha-Glucanotransferase (GTase) is an essential enzyme in alpha-1,4-glucan metabolism in bacteria and plants. It catalyses the transfer of maltooligosaccharides from an 1,4-alpha-D-glucan molecule to the 4-hydroxyl group of an acceptor sugar molecule. The crystal structures of Thermotoga maritima GTase and its complex with the inhibitor acarbose have been determined at 2.6A and 2.5A resolution, respectively. The GTase structure consists of three domains, an N-terminal domain with the (beta/alpha)(8) barrel topology (domain A), a 65 residue domain, domain B, inserted between strand beta3 and helix alpha6 of the barrel, and a C-terminal domain, domain C, which forms an antiparallel beta-structure. Analysis of the complex of GTase with acarbose has revealed the locations of five sugar-binding subsites (-2 to +3) in the active-site cleft lying between domain B and the C-terminal end of the (beta/alpha)(8) barrel. The structure of GTase closely resembles the family 13 glycoside hydrolases and conservation of key catalytic residues previously identified for this family is consistent with a double-displacement catalytic mechanism for this enzyme. A distinguishing feature of GTase is a pair of tryptophan residues, W131 and W218, which, upon the carbohydrate inhibitor binding, form a remarkable aromatic "clamp" that captures the sugar rings at the acceptor-binding sites +1 and +2. Analysis of the structure of the complex shows that sugar residues occupying subsites from -2 to +2 engage in extensive interactions with the protein, whereas the +3 glucosyl residue makes relatively few contacts with the enzyme. Thus, the structure suggests that four subsites, from -2 to +2, play the dominant role in enzyme-substrate recognition, consistent with the observation that the smallest donor for T.maritima GTase is maltotetraose, the smallest chain transferred is a maltosyl unit and that the smallest residual fragment after transfer is maltose. A close similarity between the structures of GTase and oligo-1,6-glucosidase has allowed the structural features that determine differences in substrate specificity of these two enzymes to be analysed.     

High-resolution structure of shikimate dehydrogenase from Thermotoga maritima reveals a tightly closed conformation

Shikimate dehydrogenase (SDH), which catalyses the NADPH-dependent reduction of 3-dehydroshikimate to shikimate in the shikimate pathway, is an attractive target for the development of herbicides and antimicrobial agents. Structural analysis of a SDH from Thermotoga maritima encoded by the Tm0346 gene was performed to facilitate further structural comparisons between the various shikimate dehydrogenases. The crystal structure of SDH from T. maritima was determined at 1.45 Å by molecular replacement. SDH from T. maritima showed a monomeric architecture. The overall structure of SDH from T. maritima comprises the N-terminal α/β sandwich domain for substrate binding and the C-terminal domain for NADP binding. When the T. maritima SDH structure was compared with those of the SDHs from other species, the SDH from T. maritima was in a tightly closed conformation, which should be open for catalysis. Notably, α7 moves toward the active site (∼5 Å), which forces the SDH of T. maritima in a more closed form. Four ammonium sulfate (AMS) ions were identified in the structure. They were located in the active site and appeared to mimic the role of the substrate in terms of the enzyme activity and stability. The new high resolution structural information reported in this study, including the AMS binding sites as a potent inhibitor binding site of SDHs, is expected to supplement the existing structural data and will be useful for structure-based antibacterial discovery against SDHs.     

Substrate recognition ability differs among various prokaryotic tRNase Zs

There exists a significant difference in pre-tRNA preference among prokaryotic tRNase Zs. This is an enigma, because pre-tRNAs should form the common L-shaped structure and tRNase Zs should form the common structure based on the alphabeta/betaalpha-fold. To address this issue, we examined six different eubacterial and archaeal tRNase Zs including two newly isolated tRNase Zs for cleavage of 18 different pre-tRNA substrates. Two Thermotoga maritima, one Thermus thermophilus, one Bacillus subtilis, one Thermoplasma acidophilum, and one Pyrobaculum aerophilum enzymes were tested. To our surprise, the newly isolated proteins T. maritima and T. thermophilus showed the weak tRNase Z activity, even though their primary amino acid sequences are, on the whole, quite different from those of the typical tRNase Zs. We confirmed that substrate recognition ability is quite different among those tRNase Zs. In addition, we found that the optimal conditions as a whole differ significantly among the enzymes. From these results, we provided several clues to solve the enigma by showing the potential importance of the 74th-76th nucleotide sequence of pre-tRNA, the flexible arm length of tRNase Z, the divalent metal ion species, and the histidine corresponding His222 in T. maritima tRNase Z.