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.
Viard
T
T
Laboratoire d'Enzymologie des Acides Nucléiques, Institut de Génétique et Microbiologie, UMR 8621 CNRS, Bâtiment 400, Université de Paris Sud, Centre d'Orsay, 91405 Orsay Cedex, France.
Lamour
V
V
Duguet
M
M
Bouthier de la Tour
C
C
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003596:Cytosine / Q000378:metabolism; D017931:DNA Primers; D004264:DNA Topoisomerases, Type I / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D018105:DNA, Kinetoplast / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D020124:Thermotoga maritima / Q000201:enzymology*; D015032:Zinc / Q000378:metabolism*
D017931:DNA Primers; D018105:DNA, Kinetoplast; D003596:Cytosine; D004264:DNA Topoisomerases, Type I; D015032:Zinc