Topological characterization and modeling of the 3D structure of lipase from Pseudomonas aeruginosa.
Lipase from Pseudomonas aeruginosa is a M(r) 29 kDa protein with a single functional disulfide bond as shown by a shift in electrophoretic mobility after treatment with dithiothreitol and iodoacetamide. Limited proteolysis of lipase with Staphylococcus aureus protease V8 resulted in cleavage after amino acid residues Asp38 and Glu46. 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 alpha/beta hydrolase fold, thereby allowing us to construct a 3D model which fitted the biochemical properties. The model predicts a catalytic triad consisting of Ser82, Asp229 and His251, and contains a disulfide bond connecting residues Cys183 and Cys235. Residues Asp38 and Glu46 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 disulfide bond could take place. A striking prediction from the model was the lack of a lid-like alpha-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.
Jaeger
K E
KE
Lehrstuhl Biologie der Mikroorganismen, Ruhr-Universität, Bochum, Germany.
Ransac
S
S
Koch
H B
HB
Ferrato
F
F
Dijkstra
B W
BW
D000595:Amino Acid Sequence; D000818:Animals; D003198:Computer Simulation; D004220:Disulfides / Q000032:analysis; D004789:Enzyme Activation; D006801:Humans; D006868:Hydrolysis; D008049:Lipase / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D004220:Disulfides; D008049:Lipase