Korormicin insensitivity in Vibrio alginolyticus is correlated with a single point mutation of Gly-140 in the NqrB subunit of the Na(+)-translocating NADH-quinone reductase.
Na(+)-translocating NADH-quinone reductase (NQR) from the marine Vibrio alginolyticus is strongly inhibited by a new antibiotic korormicin. Korormicin specifically inhibits the Na(+)-dependent reaction of the NQR complex and acts as a purely non-competitive inhibitor for Q-1 with the inhibitor constant of 82 pM. Korormicin-resistant mutants were isolated from V. alginolyticus and the NQR complex was purified from a mutant KR2. Similar to 2-n-heptyl-4-hydroxyquinoline N-oxide (HQNO), korormicin acted as a purely noncompetitive inhibitor to the NQR complex from the mutant KR2, but the inhibitor constant increased to 8 microM, which is 10(5)-fold higher than that of the wild-type NQR complex. The inhibitor constant of HQNO, however, was only slightly affected by the acquisition of korormicin resistance. The spontaneous mutation was caused by a single mutation of G-422 to T-422 in the nucleotide sequence of the nqrB gene, which resulted in the conversion of Gly-140 to Val-140. Thus, Gly-140 seems to play an important role for the binding of korormicin to the NqrB subunit. The fact that korormicin is a purely noncompetitive inhibitor for Q-1 strongly supports the presence of one of Q-1 binding sites in the NqrB subunit, which also has a covalently bound FMN at Thr-235.
Hayashi
Maki
M
Graduate School of Pharmaceutical Sciences, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522, Japan. makiha@p.chiba-u.ac.jp
Shibata
Naoaki
N
Nakayama
Yuji
Y
Yoshikawa
Kazuhiro
K
Unemoto
Tsutomu
T
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000494:pharmacology*; D001426:Bacterial Proteins; D001665:Binding Sites / Q000235:genetics; D024881:Drug Resistance, Bacterial / Q000235:genetics; D005231:Fatty Acids, Unsaturated / Q000737:chemistry / Q000494:pharmacology*; D005798:Genes, Bacterial; D005998:Glycine / Q000737:chemistry; D006912:Hydroxyquinolines / Q000737:chemistry / Q000494:pharmacology; D007783:Lactones / Q000737:chemistry / Q000494:pharmacology*; D008969:Molecular Sequence Data; D017354:Point Mutation; D021122:Protein Subunits; D011808:Quinone Reductases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics*; D014733:Vibrio / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics*
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D005231:Fatty Acids, Unsaturated; D006912:Hydroxyquinolines; D007783:Lactones; D021122:Protein Subunits; C111296:korormicin; C001333:2-(n-heptyl)-4-hydroxyquinoline N-oxide; C084142:sodium-translocating NADH-quinone reductase; D011808:Quinone Reductases; D005998:Glycine