Isoleucine 10 is essential for DNA gyrase B function in Escherichia coli.
DNA gyrase is an essential enzyme that regulates the DNA topology in bacteria. It belongs to the type II DNA topoisomerase family and is responsible for the introduction of negative supercoils into DNA at the expense of hydrolysis of ATP molecules. The aim of the present work was to study the contribution of I10, one of the most important residues responsible for the stabilization of GyrB dimer and involved in the ATP-binding step, in the ATP-hydrolysis reaction and in the DNA supercoiling mechanism. We constructed MBP-tagged GyrB mutants I10G and Delta4-14. Our results demonstrate that both mutations severely affect the DNA-dependent ATPase activity and DNA supercoiling. Mutation of Y5 residue involved in the formation of ATPase catalytic site (Y5G mutant) had only little effect on the DNA-dependent ATPase activity and DNA supercoiling. Interestingly, the DNA-relaxation activity of MBP-GyrB mutants and wild type was completely inhibited by ATP. Binding of ADPNP to MBP-tagged mutants was significantly decreased. ADPNP had no effect on 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 efficiency and DNA transfer through DNA gyrase.
Brino
L
L
Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), CNRS/INSERM, Université Louis-Pasteur, B.P. 163, 67404 Illkirch, cedex France.
Bronner
C
C
Oudet
P
P
Mousli
M
M
D000255:Adenosine Triphosphate / Q000378:metabolism; D020134:Catalytic Domain / Q000235:genetics; D003001:Cloning, Molecular; D027081:DNA Gyrase; D004250:DNA Topoisomerases, Type II / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005798:Genes, Bacterial; D007532:Isoleucine / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000235:genetics
D007532:Isoleucine; D000255:Adenosine Triphosphate; D027081:DNA Gyrase; D004250:DNA Topoisomerases, Type II