DNA gyrase requires DNA for effective two-site coordination of divalent metal ions: further insight into the mechanism of enzyme action.
The catalytic properties of DNA gyrase, an A 2B 2 complex, are modulated by the presence of divalent metal ions. Using circular dichroism, protein melting experiments and enzyme activity assays, we investigated the correlation between the A 2B 2 conformation, the nature of the metal ion cofactor and the enzyme activity in the presence and absence of DNA substrate. At room temperature, DNA gyrase structure is not appreciably affected by Ca (2+) or Mg (2+) but is modified by Mn (2+). In addition, metal ions strongly affect the enzyme's thermal transitions, rendering the A 2B 2 structure more flexible. Using the B subunit, we were able to identify two distinct complexes with manganese ions. The first one exhibits a 1:1 stoichiometry and is not affected by the presence of DNA. The second complex is associated with a large protein structural modification that can be remarkably modulated by addition of the DNA substrate. This behavior is conserved in the reconstituted protein. Studies with two GyrB mutants indicate that Mn (2+) interference with the TOPRIM region modulates gyrase supercoiling activity. In particular, considering the need for two divalent metal ions for an efficient catalytic cleavage of the phosphodiester bond, our data suggest that residue D500 participates in the first complexation event (DNA-independent), whereas residue D498 is involved mainly in the second process. In conclusion, a combination of the ion features (ionic size, electronegativity, coordination sphere) operating at the level of the catalytic region and of the ion-driven modifications in overall enzyme structure and flexibility contribute to the mechanism of gyrase activity. An effectual role for DNA recruiting the second catalytic metal ion is envisaged.
Sissi
Claudia
C
Department of Pharmaceutical Sciences, University of Padova, Via Marzolo, 5, 35131 Padova, Italy.
Chemello
Angelita
A
Vazquez
Elena
E
Mitchenall
Lesley A
LA
Maxwell
Anthony
A
Palumbo
Manlio
M
D004247:DNA / Q000378:metabolism*; D027081:DNA Gyrase / Q000378:metabolism*; D008274:Magnesium / Q000737:chemistry*; D008345:Manganese / Q000737:chemistry*; D011487:Protein Conformation; D015032:Zinc / Q000737:chemistry*
D008345:Manganese; D004247:DNA; D027081:DNA Gyrase; D008274:Magnesium; D015032:Zinc