Open AccessCharacterization of the ATPase activity of the N‐terminal nucleotide binding domain of an ABC transporter involved in oleandomycin secretion by Streptomyces antibioticus
Author(s) -
Aparicio Gerardo,
Buche André,
Méndez Carmen,
Salas JoséA.
Publication year - 1996
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.1996.tb08378.x
Subject(s) - oleandomycin , biochemistry , atpase , atp binding cassette transporter , biology , fusion protein , nucleotide , enzyme , atp hydrolysis , chemistry , escherichia coli , transporter , recombinant dna , gene , erythromycin , antibiotics
The ole B gene of Streptomyces antibioticus , oleandomycin producer, encodes an ABC transporter containing two putative ATP‐binding domains and is involved in oleandomycin resistance and secretion in this organism. We have overexpressed in Escherichia coli the N‐terminal nucleotide‐binding domain of OleB (OleB′) as a fusion protein to a maltose‐binding protein and purified the fusion protein by affinity chromatography. The fusion protein showed ATPase activity dependent on the presence of Mg 2+ ions. ATPase activity was resistant to specific inhibitors of P‐, F‐, and V‐type ATPase whereas sodium azide and 7‐chloro‐4‐nitrobenzo‐2‐oxa‐l,3‐diazole (NBD‐C1) were strong inhibitors. The change of Lys71, located within the Walker A motif of the OleB′ protein, to Gin or Glu caused a loss of ATPase activity, whereas changing to Gly did not impair the activity. The results suggest that the intrinsic ATPase activity of purified fusion protein can be clearly distinguished from other ATP‐hydrolysing enzymes, including ion‐translocating ATPases or ABC‐traffic ATPases, both on the basis of inhibition by different agents and since it hydrolyzes ATP without interacting with a hydrophobic membrane component.