Isoleucine 10 is essential for DNA gyrase B function in Escherichia coli

DNA gyrase is an essential enzyme that regulates the DNA topology in bacter ia. It belongs to the type II DNA topoisomerase family and is responsible f or the introduction of negative supercoils into DNA at the expense of hydro lysis of ATP molecules. The aim of the present work was to study the contri bution of I10, one of the most important residues responsible for the stabi lization of GyrB dimer and involved in the ATP-binding step, in the ATP-hyd rolysis reaction and in the DNA supercoiling mechanism. We constructed MBP- tagged GyrB mutants I10G and Delta 4-14. Our results demonstrate that both mutations severely affect the DNA-dependent ATPase activity and DNA superco iling. Mutation of Y5 residue involved in the formation of ATPase catalytic site (Y5G mutant) had only little effect on the DNA-dependent ATPase activ ity and DNA supercoiling. Interestingly, the DNA-relaxation activity of MBP -GyrB mutants and wild type was completely inhibited by ATP. Binding of ADP NP to MBP-tagged mutants was significantly decreased. ADPNP had no effect o n DNA-relaxation activity of MBP-tagged mutants but was able to inhibit MBP -tagged wild type enzyme. Our results demonstrate that GyrB N-terminal arm, and specially I10 residue is essential for ATP binding/hydrolysis efficien cy and DNA transfer through DNA gyrase. (C) Societe francaise de biochimie et biologie moleculaire / Editions scientifiques et medicales Elsevier SAS.