Specificity of the lipase-specific foldases of gram-negative bacteria and the role of the membrane anchor

Abstract

Folding of lipases that are secreted by Pseudomonads and other gram-negative bacteria via the type II secretion pathway is facilitated by dedicated chaperones, called lipase-specific foldases (Lifs). Lifs are membrane-anchored proteins with a large periplasmic domain. The functional interaction between the Lif and its cognate lipase is specific, since the Pseudomonas aeruginosa Lif was found not to substitute for Lifs from Burkholderia glumae or Acinetobacter calcoaceticus. However, the P. aeruginosa Lif was able to activate the lipase from the closely related species P. alcaligenes. Hybrid proteins constructed from parts of the P. aeruginosa and B. glumae Lifs revealed that the C-terminal 138 amino acids of the B. glumae Lif determine the specificity of the interaction with the cognate lipase. Furthermore, the periplasmic domain of the B. glumae Lif was functional when cloned in frame with a cleavable signal sequence, which demonstrates that the membrane anchor is not essential for Lif function in vivo. However, the recombinant Lif was released into the medium, indicating that the function of the membrane anchor is to prevent secretion of the Lif together with the lipase.

Secreted Enzymatic Activities of Wild-Type and pilD -Deficient Legionella pneumophila

“…The exoenzymes previously found lacking from pilD or other type II secretion mutants include the following: the esterase and lipase of Acinetobacter calcoaceticus; the acyltransferase, aerolysin, and protease of A. hydrophila; the lipases of Burkholderia sp. ; the pectate lyase and cellulase of Erwinia chrysanthemi; the pullulanase of Klebsiella oxytoca; the alkaline phosphatase, lipase, elastase, exotoxin A, LasA protease, and PLC of P. aeruginosa; the cholera toxin, protease, and endochitinase of V. cholerae; and the amylase, cellulase, endoglucanase, and protease of Xanthomonas campestris (14,20,24,33,36,44,55,56,58,63,70). Thus, there is precedent for a linkage between pilD and an esterase-lipase and protease.…”

“…First, supernatants were assayed for the release of pNP from p-nitrophenyl caprylate and p-nitrophenyl palmitate (Sigma) (3,20,24,34,73,75). Briefly, 100 l of sample was added to 1 ml of buffer (i.e., 100 mM Tris [pH 8] and 0.2% Triton X-100) containing 1 mM of substrate, and then the increase in absorbance at 410 nm was measured after 5 and 15 min of incubation at 37°C.…”

Influence of Deletions within Domain II of Exotoxin A on Its Extracellular Secretion from Pseudomonas aeruginosa

“…The proteins are subsequently released into the periplasm, where they fold into what appears to be their final conformation. Several studies have demonstrated that disulfide bond formation and chaperone-assisted folding are crucial steps in the generation of a protein competent for the final step in the secretion process (2,3,7,31). During this step, the folded mature proteins are translocated across the outer membrane via a specialized machinery called Xcp in P. aeruginosa, i.e., the main terminal branch of the general secretory pathway, or type II pathway (10).…”

Lipase and Its Modulator from Pseudomonas sp. Strain KFCC 10818: Proline-to-Glutamine Substitution at Position 112 Induces Formation of Enzymatically Active Lipase in the Absence of the Modulator

“…In addition, the addition of the crude extract of E. coli JM109 harboring pACTM12 to the crude extract of E. coli JM109 harboring pKL11 induced the activation of the inactive lipase (data not shown). These results suggest that, despite its low sequence homology with Pseudomonas Lifs, LimK may interact directly with lipase and be required for its correct folding in the same manner that Lifs interact with lipase (9,13,17,30).…”