Glutathione as an essential factor for chaperon-mediated activation of lactonizing lipase (LipL) from Pseudomonas sp. 109.
Pseudomonas sp. 109 produces a unique lipase (LipL) which efficiently catalyzes intramolecular transesterification of omega-hydroxyesters to form macrocyclic lactones. The production of the enzymatically active LipL requires a specific molecular chaperon (LimL protein) together with a low-M(r) lipase-activation-factor (LAF) of unknown structure. From 50 g of Pseudomonas cells, 2.15 mg of LAF was purified as a sulfobenzofurazanyl derivative after methanol extraction, derivatization, and C(18) reverse-phase HPLC. One-dimensional and two-dimensional 600 MHz (1)H-NMR and fast atom bombardment mass spectrometry (FAB-MS) revealed that LAF is glutathione. Because several SH compounds (L-cysteine and mercaptoethanol) were similarly effective to native LAF in the activation of LipL, and because only LipL contains two cysteinyl residues forming an intramolecular disulfide bond, it is concluded that the reduction of and reformation of the intramolecular disulfide bond of LipL is essential to liberate free and fully active LipL.
Tanaka
J
J
Department of Biotechnology, Graduate School of Engineering, Osaka University, Suita, Osaka 565-0871, Japan.
Nihira
T
T
Yamada
Y
Y
D001426:Bacterial Proteins / Q000378:metabolism*; D004220:Disulfides / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D005464:Fluorobenzenes / Q000378:metabolism; D005978:Glutathione / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D008049:Lipase / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008565:Membrane Proteins; D018832:Molecular Chaperones / Q000378:metabolism*; D011549:Pseudomonas / Q000737:chemistry / Q000201:enzymology*; D011994:Recombinant Proteins
D001426:Bacterial Proteins; D004220:Disulfides; D005464:Fluorobenzenes; C092194:LimL protein, Pseudomonas; D008565:Membrane Proteins; D018832:Molecular Chaperones; D011994:Recombinant Proteins; C037802:7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate; D008049:Lipase; C120013:lactonizing lipase protein, Pseudomonas; D005978:Glutathione