Disulfide Bond in Pseudomonas aeruginosa Lipase Stabilizes the Structure but Is Not Required for Interaction with Its Foldase

Abstract

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...

DsbA and DsbC Are Required for Secretion of Pertussis Toxin by Bordetella pertussis

“…For example, E. coli type IV pilin, the pullulanase secreton pilot protein PulS, and P. aeruginosa lipase all require correct disulfide formation for stability (29,36,50). The absence of the major periplasmic oxidant, DsbA, was shown to result in instability of both S1 and S2.…”

“…Periplasmic DsbC promotes disulfide bond exchange (which is often needed for proteins that possess more than one disulfide bond) in previously oxidized proteins (37,48). Dsb proteins have been shown to be essential for correct folding or assembly of a number of proteins and proteinaceous complexes, including enteropathogenic E. coli type IV pili (50), the E. coli flagellar apparatus (13), the Klebsiella oxytoca type II secreton (36), the E. coli PapD P-pilus chaperone (24), Pseudomonas aeruginosa lipase (LipA) (29,41), E. coli heat-labile toxin (47), and cholera toxin (32,47).…”

A Short Review on Various Screening Methods to Isolate Potential Lipase Producers: Lipases-the Present and Future Enzymes of Biotech Industry

“…Spectrophotometric methods using pNP napthol and thiol esters: These methods mainly involve use of various synthetic lipidic substrates like p-nitrophenyl esters of the long chain fatty acids which upon enzyme hydrolysis transforms into yellow colored p-nitrophenol and is measured at 405-410 nm (Huggins and Lapides, 1947;Stuer et al, 1986;Kojima et al, 1994;Ushio et al, 1996;Becker et al, 1997;Liebeton et al, 2001). One unit (U) of lipase activity was defined as the amount of enzyme that released 1 μm of p-nitrophenol minG 1 under the standard assay conditions.…”

Hexadecane and Tween 80 Stimulate Lipase Production in Burkholderia glumae by Different Mechanisms

“…Folding of lipase is facilitated by the specific intermolecular chaperone Lif (Lipase-specific foldase) (31), which is encoded by the gene designated lipB in the case of Burkholderia glumae (8) and by general folding catalysts, such as DsbA and DsbC (39), which catalyze the formation and isomerization of disulfide bonds. The single disulfide bond in lipase and a bound Ca 2ϩ ion are not necessary for activity but provide stability to the protein under harsh conditions (6,23). Enzymatically active lipase is then secreted across the outer membrane into the extracellular medium via the type II secretion pathway (38).…”