Abstract
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.
Highlights
Topoisomerases, by controlling DNA supercoiling state, are key enzymes for adaptation to high temperatures in thermophilic organisms
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
We have focused on the study of the T. maritima topoisomerase I overexpressed in E. coli and used mutational analysis to explore some of its biochemical properties
Summary
In mesophilic bacteria (i.e. Escherichia coli), the DNA supercoiling control is achieved mainly through the balance between negative supercoiling impulsed by gyrases and DNA relaxation by topoisomerase I [1, 2] In hyperthermophilic organisms, this control appears important for life at high temperatures. Examining the C-terminal part of the protein including the last 93 amino acids, no similarity with the other topoisomerases of the family was found [4], except a putative zinc-binding motif with the structure Cys-X-Cys-X16-Cys-X-Cys (Fig. 1) This type of tetracysteine motif is present in the C-terminal part of most bacterial topoisomerases I sequences with a variable number of copies of up to four. The function of the zinc motif in T. maritima topoisomerase I remains unknown, but our results support the idea that the mechanism of the hyperthermophilic topoisomerase I may be somewhat different from that of its E. coli mesophilic homolog
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