Pseudomonas aeruginosa secretes a 29-kDa lipase which is dependent for folding 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 severely reduced levels of extracellular lipase activity, indicating the importance of the disulfide bond for secretion of lipase through the outer membrane. Wild-type and variant lipase genes fused to the signal sequence of pectate lyase from Erwinia carotovora were expressed in Escherichia coli, denatured by treatment with urea, and subsequently refolded in vitro. Enzymatically active lipase was obtained irrespective of the presence or absence of the disulfide bond, suggesting that the disulfide bond is required neither for correct folding nor for the interaction with the lipase-specific foldase. However, cysteine-to-serine variants were more readily denatured by treatment at elevated temperatures and more susceptible to proteolytic degradation by cell lysates of P. aeruginosa. These results indicate a stabilizing function of the disulfide bond for the active conformation of lipase. This conclusion was supported by the finding that the disulfide bond function could partly be substituted by a salt bridge constructed by changing the two cysteine residues to arginine and aspartate, respectively.The gram-negative bacterium Pseudomonas aeruginosa secretes an array of different enzymes via three distinct secretion pathways (40,42,43,46). Most of them are exported in two consecutive steps through the so-called type II secretory pathway into the extracellular medium (11), among them a lipase LipA with a molecular mass of 29 kDa (39). This lipase shows remarkable enzymatic characteristics, e.g., a pronounced stereoselectivity (32) and a broad substrate specificity (23), making it an interesting candidate for biotechnological applications (24).Enzymes being secreted via the type II secretion pathway contain a N-terminal signal sequence mediating the translocation across the cytoplasmic membrane by the Sec apparatus which has intensively been studied in Escherichia coli (8,31). An additional machinery consisting of at least 12 so-called Xcp proteins is required for the secretion of the periplasmic intermediates through the outer membrane of P. aeruginosa (11). Although much effort has been expended to identify a potential recognition signal for the Xcp machinery within the extracellular proteins which distinguishes them from periplasmic proteins, no common linear recognition sequence has so far been identified. There is increasing evidence that extracellular proteins fold into a transport-competent conformation before translocation across the outer membrane (3,5,(12)(13)(14)(15)30), suggesting that the formation necessary for secretion is a threedimensional structural element composed of noncontiguous amino acid residues or parts of the primary structure. Many of the proteins secreted via the type II pathway contain a disulfide bon...