Characterization of a novel enoyl-acyl carrier protein reductase of diazaborine-resistant Rhodobacter sphaeroides mutant.
Rhodobacter sphaeroides contains two enoyl-acyl carrier protein (ACP) reductases, FabI(1) and FabI(2). However, FabI(1) displays most of the cellular enzyme activity. The spontaneous diazaborine-resistant mutation was mapped as substitution of glutamine for proline 155 (P155Q) of FabI(1). The mutation of FabI(1)[P155Q] increased the specificity constants (k(cat)/K(m)) for crotonyl-ACP and NADH by more than 2-fold, while the site-directed mutation G95S (FabI(1)[G95S]), corresponding to the well-known G93 mutation of Escherichia coli FabI, rather decreased the values. Inhibition kinetics of the enzymes revealed that triclosan binds to the enzyme in the presence of NAD(+), while the diazaborine appears to interact with NADH and NAD(+) in the enzyme active site. The apparent inhibition constant K(i)(') of triclosan for FabI(1)[P155Q] and FabI(1)[G95S] at saturating NAD(+) were approximately 80- and 3-fold higher than that for the wild-type enzyme, respectively, implying that the inhibition was remarkably impaired by the P155Q mutation. The similar levels of K(i)(') of diazaborine for the mutant enzymes were also observed with respect to NAD(+). Thus, the novel mutation P155Q appears to disturb the binding of inhibitors to the enzyme without affecting the catalytic efficiency.
Lee
Il-Han
IH
Department of Life Science, Sogang University, Mapo, Shinsu 1, Seoul 121-742, South Korea.
Kim
Eui-Jin
EJ
Cho
Yoo-Hee
YH
Lee
Jeong K
JK
D000595:Amino Acid Sequence; D000891:Anti-Infective Agents, Local / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D001896:Boron Compounds / Q000494:pharmacology*; D024881:Drug Resistance, Bacterial / Q000235:genetics; D050753:Enoyl-(Acyl-Carrier-Protein) Reductase (NADH); D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017354:Point Mutation; D012242:Rhodobacter sphaeroides / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D016415:Sequence Alignment; D014260:Triclosan / Q000494:pharmacology
D000891:Anti-Infective Agents, Local; D001426:Bacterial Proteins; D001896:Boron Compounds; D007527:Isoenzymes; D009243:NAD; D014260:Triclosan; D010088:Oxidoreductases; D050753:Enoyl-(Acyl-Carrier-Protein) Reductase (NADH)