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 catal yzes intramolecular transesterification of omega-hydroxyesters to form macr ocyclic 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 cel ls, 2.15 mg of LAF was purified as a sulfobenzofurazanyl derivative after m ethanol extraction, derivatization, and C-18 reverse-phase HPLC. one-dimens ional and two-dimensional 600 MHz H-1-NMR and fast atom bombardment mass sp ectrometry (FAB-MS) revealed that LAF is glutathione. Because several SH co mpounds (L-cysteine and mercaptoethanol) were similarly effective to native LAF in the activation of LipL, and because only LipL contains two cysteiny l residues forming an intramolecular disulfide bond, it is concluded that t he reduction of and reformation of the intramolecular disulfide bond of Lip L is essential to liberate free and fully active LipL.