Topological characterization and modeling of the 3D structure of lipase from Pseudomonas aeruginosa

Abstract

Lipase from &x4domonas ueruginosu is a M, 29 kDa protein with a single functional disultide bond as shown by a shift in electrophoretic mobility after treatment with dithiothreitol and iodoacetamide. Liited proteolysis of lipase with Staphylococcus uureus protease V8 resulted in cleavage after amino acid residues Asp)' and GIu~. Comparison of the lipase amino acid sequence with those of other hydrolases with known 3D structures indicated that the folding pattern might be compatible with the al/l hydrolase fold, thereby allowing us to construct a 3D model which fitted the biochemical properties. The model predicts a catalytic triad consisting of Se?, Asp*" and Hi?', and contains a disuhide bond connecting residues Cys'S) and C~S*~~. Residues Asp3* and Gl@ are located at the surface of the enzyme, whereas the disulfide bond is rather inaccessible, which is in agreement with the finding that the protein needed to be partly unfolded before a reduction of the disultide bond could take place. A striking prediction from the model was the lack of a lid-like a-helical loop structure covering the active site which confers to other well-characterized lipases a unique property known as interfacial activation. Experimental determination of lipase activity under conditions where the substrate existed either as monomeric solutions or aggregates confirmed the absence of interfacial activation.

The crystal structure of Bacillus subtili lipase: a minimal α/β hydrolase fold enzyme

“…On the basis of a multiple sequence alignment of several microbial lipases, Ser77 was suggested to be the nucleophile in B. subtilis lipase 6 and Asp133 was proposed as the catalytic acid. 21 The S77C and D133N mutants were indeed devoid of lipase activity. 22 For the active-site histidine residue, two candidates were present initially, His152 and His156.…”

Partial Interfacial Activation ofProteus vulgarisLipase Overexpressed inEscherichia coli

“…3b, P. vulgaris lipase showed a partial interfacial activation, indicating that this enzyme is able to degrade both PNPB emulsions and monomeric PNPB,8) while HLL and CRL degrade only one form of substrate, either PNPB emulsions or monomeric PNPB. Partial interfacial activation is a new category found in certain lipases (from P. vulgaris and P. aeruginosa 9 ) different from any types so far reported.…”

Phospholipase PlaB of Legionella pneumophila Represents a Novel Lipase Family

“…Lipases mostly share a common ␣/␤-hydrolase folding pattern, at which conserved motifs surround the nucleophilic residue (Ser, Asp, or Cys), the catalytic acid (Asp or Glu) and histidine (33). However, PlaB and homologs differ from previously published enzymes, since they do not exhibit a GXSXG or a GDSL motif, characteristically displayed by eukaryotic and prokaryotic lipases and esterases as well (12,34). Instead, PlaB constitutes the designated motif THSTG, surrounding the catalytically active serine.…”

“…Alignment to PlaB homologs displays His-7 to be as conserved as residues constituting the catalytic triad. Adjacent to a central glycine, which most likely represents a part of the oxyanion hole, His-7 probably facilitates hydrolysis of deduced substrates through stabilization of the tetrahedral intermediate (34). Regarding conservation of Ser-129, PlaB-like proteins of Psychromonas or Desulfuromonas exhibit a Gln residue at the designated position.…”