Disulfide bond in Pseudomonas aeruginosa lipase stabilizes the structure but is not required for interaction with its foldase

Pseudomonas aeruginosa secretes a 29-kDa lipase which is dependent for fold ing on the presence of the lipase-specific foldase Lif, The lipase contains two cysteine residues which form an intramolecular disulfide bond, Variant lipases with either one or both cysteines replaced by serines showed sever ely reduced levels of extracellular lipase activity, indicating the importa nce of the disulfide bond for secretion of lipase through the outer membran e, Wild-type and variant lipase genes fused to the signal sequence of pecta te lyase from Erwinia carotovora were expressed in Escherichia coli, denatu red by treatment with urea, and subsequently refolded in vitro. Enzymatical ly active lipase was obtained irrespective of the presence or absence of th e disulfide bond, suggesting that the disulfide bond is required neither fo r correct folding nor for the interaction with the lipase-specific foldase, However, cysteine-to-serine variants were more readily denatured by treatm ent at elevated temperatures and more susceptible to proteolytic degradatio n by cell lysates of P, aeruginosa. These results indicate a stabilizing fu nction of the disulfide bond for the active conformation of lipase, This co nclusion was supported by the finding that the disulfide bond function coul d partly be substituted by a salt bridge constructed by changing the two cy steine residues to arginine and aspartate, respectively.