N-terminal transmembrane domain of lipase LipA from Pseudomonas protegens Pf-5: A must for its efficient folding into an active conformation.

LipA from Pseudomonas protegens Pf-5 has been proven not to be secreted into the extracytoplasmic space, proposing that it is a membrane protein in virtue of its N-terminal transmembrane domain predicted by the TMHMM 2.0. However, LipA was confirmed to be an intracellular protein through determining the effects of lipA deletion or overexpression on the lipase activities in the whole-cell, lysis supernatant and lysis pellet, even through its transmembrane domain being able to make heterologous LacZ locate on the cytoplasmic membrane via construction of β-galactosidase reporter strains. Subsequently, lipase activity assays showed that the transmembrane domain played an indispensable role for the catalytic function of LipA through construction of the markerless deletion mutant of transmembrane domain sequence of lipA and the expression and purification of LipA and LipAΔTMD. To further investigate why the transmembrane domain lost its membrane localization function and significantly affected the catalytic function of LipA, the 3D structures of LipA and LipAΔTMD were constructed. The results indicated that the transmembrane domain, located in the interior of LipA, helped the α-helical lid to form an open conformation by the mediation of α5 helix. It seems to act as a kind of intramolecular chaperone like the β-roll motif of subfamily I.3 lipases, which is novel and is the first to notify the intramolecular chaperone of a subfamily I.1 lipase.