Hyperthermophilic topoisomerase I from Thermotoga maritima. A very efficient enzyme that functions independently of zinc binding.

Topoisomerases, by controlling DNA supercoiling state, are key enzymes for adaptation to high temperatures in thermophilic organisms. We focus here on the topoisomerase I from the hyperthermophilic bacterium Thermotoga maritima (optimal growth temperature, 80 degrees C). To determine the properties of the enzyme compared with those of its mesophilic homologs, we overexpressed T. maritima topoisomerase I in Escherichia coli and purified it to near homogeneity. We show that T. maritima topoisomerase I exhibits a very high DNA relaxing activity. Mapping of the cleavage sites on a variety of single-stranded oligonucleotides indicates a strong preference for a cytosine at position -4 of the cleavage, a property shared by E. coli topoisomerase I and archaeal reverse gyrases. As expected, the mutation of the putative active site Tyr 288 to Phe led to a totally inactive protein. To investigate the role of the unique zinc motif (Cys-X-Cys-X(16)-Cys-X-Cys) present in T. maritima topoisomerase I, experiments have been performed with the protein mutated on the tetracysteine motif. Strikingly, the results show that zinc binding is not required for DNA relaxation activity, contrary to the E. coli enzyme. Furthermore, neither thermostability nor cleavage specificity is altered in this mutant. This finding opens the question of the role of the zinc-binding motif in T. maritima topoisomerase I and suggests that this hyperthermophilic topoisomerase possesses a different mechanism from its mesophilic homolog.

Reverse gyrase from the hyperthermophilic bacterium Thermotoga maritima: properties and gene structure.

The hyperthermophilic bacterium Thermotoga maritima MSB8 possesses a reverse gyrase whose enzymatic properties are very similar to those of archaeal reverse gyrases. It catalyzes the positive supercoiling of the DNA in an Mg2+- and ATP-dependent process. Its optimal temperature of activity is around 90 degrees C, and it is highly thermostable. We have cloned and DNA sequenced the corresponding gene (T. maritima topR). This is the first report describing the analysis of a gene encoding a reverse gyrase in bacteria. The T. maritima topR gene codes for a protein of 1,104 amino acids with a deduced molecular weight of 128,259, a value in agreement with that estimated from the denaturing gel electrophoresis of the purified enzyme. Like its archaeal homologs, the T. maritima reverse gyrase exhibits helicase and topoisomerase domains, and its sequence matches very well the consensus sequence for six reverse gyrases now available. Phylogenetic analysis shows that all reverse gyrases, including the T. maritima enzyme, form a very homogeneous group, distinct from the type I 5' topoisomerases of the TopA subfamily, for which we have previously isolated a representative gene in T. maritima (topA). The coexistence of these two distinct genes, coding for a reverse gyrase and an omega-like topoisomerase, respectively, together with the recent description of a gyrase in T. maritima (O. Guipaud, E. Marguet, K. M. Noll, C. Bouthier de la Tour, and P. Forterre, Proc. Natl. Acad. Sci. USA 94:10606-10611, 1977) addresses the question of the control of the supercoiling in this organism.

Reverse gyrase gene from Sulfolobus shibatae B12: gene structure, transcription unit and comparative sequence analysis of the two domains.

We cloned and sequenced a DNA fragment from the thermophilic archaeal strain Sulfolobus shibatae B12 that includes the gene topR encoding the reverse gyrase. The RNA of the reverse gyrase gene was characterized indicating that the topR gene is fully functional in vivo. We showed by primer extension analysis that transcription of topR initiates 28 bp downstream from a consensus A-box promoter. In order to understand how this particular type I DNA topoisomerase introduces positive superturns into the DNA, we compared the amino acid sequence of reverse gyrase from S.shibatae with the two other known reverse gyrases. This comparison indicates a common organization of these proteins: the carboxy-terminal domain is related to the type I-5' topoisomerase family while the amino-terminal domain possesses some motifs of proteins described as RNA or DNA helicases. By using local alignments, we showed that (i) reverse gyrases constitute a new and rather homogenous group within the type I-5' DNA topoisomerase family; (ii) a careful sequence analysis of the amino-terminal domain allows us to relate the presence of some motifs with an ATP binding and hydrolysis reaction coupled to a DNA binding and unwinding activity.

Cloning and sequencing of the gene coding for topoisomerase I from the extremely thermophilic eubacterium, Thermotoga maritima.

A 2767 bp fragment containing a gene coding for a topoisomerase I from the extremely thermophilic eubacterium Thermotoga maritima (Tm TopA) has been cloned and sequenced. The protein is composed of 633 amino acids with a calculated molecular mass of 72,695 Da. It shares significant similarity with the topoisomerases I of mesophilic eubacteria. The highest score is obtained with Bacillus subtilis (44% identity); in particular, T. maritima and B. subtilis possess an insertion of 7-8 amino acids in the vicinity of the active site, that is absent in topoisomerases of other organisms. A specific feature of T. maritima topoisomerase I is its low cysteine content compared to its mesophilic homologs. It contains 5 cysteine residues, of which 4 could constitute a zinc finger motif. Finally, analysis of the regions flanking the gene reveals that Tm TopA is surrounded by two other ORFs, suggesting the occurrence of a polycistronic transcriptional unit.

ATP-independent DNA topoisomerase from Fervidobacterium islandicum.

Thermotogales are thermophilic eubacteria belonging to a very slowly evolving branch in the eubacterial tree. In this report, we describe the purification and characterization of an ATP-independent DNA topoisomerase from the Thermotogale, Fervidobacterium islandicum. The enzyme, a monomer of about 75 kDa, is a type I DNA topoisomerase sharing many properties with the other bacterial topoisomerases I: it absolutely requires Mg2+ for activity, relaxes negatively but not positively supercoiled DNA and is inhibited by single-stranded M13 DNA and spermidine. A feature of the F. islandicum ATP-independent DNA topoisomerase I is its thermophily. The optimal temperature for the enzymatic activity is 75 degrees C. Studies about thermostability show that the enzyme is more stable when incubated undiluted in the storage buffer. In these conditions, 60% activity was retained after a 30 min preincubation at 75 degrees C.

Reverse gyrase in thermophilic eubacteria.

The presence of reverse gyrase, an unusual ATP-dependent type I topoisomerase first isolated from thermophilic archaebacteria, has been detected in four strains of Thermotogales, an order of extremely thermophilic eubacteria. This result suggests that reverse gyrase plays a key role in high-temperature-living organisms, independently of the evolutionary kingdom to which they belong.

Reverse gyrase, a hallmark of the hyperthermophilic archaebacteria.

Investigation of the presence of a reverse gyrase-like activity in archaebacteria revealed wide distribution of this activity in hyperthermophilic species, including methanogens and sulfur-dependent organisms. In contrast, no reverse gyrase activity was detected in mesophilic and moderately thermophilic organisms, which exhibited only an ATP-independent activity of DNA relaxation. These results suggest that the presence of reverse gyrase in archaebacteria is tightly linked to the high growth temperatures of these organisms. With respect to antigenic properties, the enzyme appeared similar among members of the genus Sulfolobus. In contrast, no close antigenic relatedness was found between the reverse gyrase of members of the order Sulfolobales and that of the other hyperthermophilic organisms.

Pyridoxal 5'phosphate binding site of Escherichia coli beta cystathionase and cystathionine gamma synthase comparison of their sequences.

The phosphopyridoxyl peptides of beta cystathionase and cystathionine gamma synthase of Escherichia Coli were identified after reduction, carboxymethylation and proteolysis of the holoenzymes. Their comparison with those obtained from rat liver gamma cystathionase (Fearon, C.W., Rodkey, J.A. and Abeles R.H. 1982. Biochemistry 21 3790-3794.) showed a high degree of homology between the three PLP binding sites with the presence of the tripeptide sequence: Thr-Lys(Pxy)-Tyr in their structure. This homology suggests that these enzymes of methionine metabolism have probably the same origin.