Mutations in the B subunit of Escherichia coli DNA gyrase that affect ATP-dependent reactions.
We have previously reported specific labeling of Escherichia coli DNA gyrase by the ATP affinity analog pyridoxal 5'-diphospho-5'adenosine (PLP-AMP), which resulted in inhibition of ATP-dependent reactions. The analog was found to be covalently bound at Lys103 and Lys110 on the gyrase B subunit (Tamura, J. K., and Gellert, M. (1990) J. Biol. Chem. 265, 21342-21349). In this study, the importance of these two lysine residues is examined by site-directed mutagenesis. Substitutions of Lys 103 result in the loss of ATP-dependent functions. These mutants are unable to supercoil DNA, to hydrolyze ATP, or to bind a nonhydrolysable ATP analog, 5'-adenylyl-beta,gamma-imidodiphosphate (ADPNP). The ATP-independent functions of gyrase, such as relaxation of negatively supercoiled DNA and oxolinic acid-induced cleavage of double-stranded DNA, are unaffected by these mutations, suggesting that the mutant B subunits are assembling correctly with the A subunits. Gyrase with substitutions of Lys110 retains all activities. However, the affinity of ATP is decreased. The DNA supercoiling activity of gyrase A2B2, tetramers reconstituted with varying ratios of inactive mutant and wild-type gyrase B subunits is consistent with a mechanism of DNA supercoiling that requires the interdependent activity of both B subunits in ATP binding and hydrolysis.
O'Dea
M H
MH
Laboratory of Molecular Biology, NIDDK, National Institutes of Health, Bethesda, Maryland 20892-0540, USA.
Tamura
J K
JK
Gellert
M
M
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000266:Adenylyl Imidodiphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D027081:DNA Gyrase; D004250:DNA Topoisomerases, Type II / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D017354:Point Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D000266:Adenylyl Imidodiphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D027081:DNA Gyrase; D004250:DNA Topoisomerases, Type II