Random mutagenesis on the Pseudomonas lipase activator protein, LipB: exploring amino acid residues required for its function.
LipB, lipase activator protein from Pseudomonas aeruginosa TE3285, specifically recovers the enzymatic activity of denatured inactive lipase. To find important amino acid residues of LipB in this reactivation, random mutagenesis using error-prone PCR was performed on a gene encoding the functional region of LipB. The resultant DNA library was introduced into the lipase expression system using Escherichia coli, and LipB mutants lacking lipase activity were selected by two screening procedures. First, on agar plates containing tributyrin as a substrate for lipase, single colonies lacking active lipase secretion were selected as clones missing the active LipB. Second, to exclude nonsense and frameshift mutants, the molecular size of LipB in the given clones was confirmed by Western blotting. From the selected mutants, of which multiple residues are replaced, five single-residue substituted mutants were newly prepared. Consequently, Y99C, Y99H, S102R and R115C mutants formed no detectable complex with the lipase and lost the in vitro reactivation activity. In the case of Y99C and R115C, their single cysteine residue formed the intermolecular disulfide bridge. Thus, Tyr99 and Arg115 are likely to exist on the molecular surface of LipB, and are candidates for residues that make direct interaction with the denatured lipase in the reactivation process.
Shibata
H
H
Institute for Chemical Research, Kyoto University, Uji, Japan.
Kato
H
H
Oda
J
J
D001426:Bacterial Proteins; D015153:Blotting, Western; D004789:Enzyme Activation / Q000187:drug effects; D004793:Enzyme Reactivators; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D008049:Lipase / Q000378:metabolism; D008970:Molecular Weight; D016296:Mutagenesis; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D011489:Protein Denaturation; D011550:Pseudomonas aeruginosa / Q000737:chemistry* / Q000235:genetics; D013329:Structure-Activity Relationship; D015534:Trans-Activators / Q000737:chemistry* / Q000235:genetics* / Q000494:pharmacology; D014280:Triglycerides / Q000378:metabolism
D001426:Bacterial Proteins; D004793:Enzyme Reactivators; D015534:Trans-Activators; D014280:Triglycerides; C088618:lipase activator protein, Bacteria; D008049:Lipase; C005830:tributyrin