The Interaction And Functional Analysis Of The Bacterial Lipase And Its Specific Foldase From Acinetobacter Sp.

Lipases (E.C.3.1.1.3) hydrolyze triacylglycerols into fatty acids, diacylglycerol, monoacylglycerol,and glycerol. Except for lipolytic hydrolysis, lipases are able to perform a wide range of bioconversions,such as interesterification, esterification, alcoholysis, acidolysis and aminolysis. Due to their prevalenceand ability to catalyze various reactions microbial lipases have been used in many important studies.The substrate specificities, thermostability, and alkaline stability of microbial lipases enable theseenzymes to be used in a wide variety of industrial applications including detergents, oil processing,cosmetics, medicine, and food. The lipase gene regulation is very strict, which limits the improvementof the fermentation level of lipase-producting strains, and many lipase genes fail to be expressed in afunctional form, instead of forming inclusion bodies. The lipases folding reseaches could pave the wayto improve the product efficiency. In this study, we focus on the function and protein interaction oflipase specific foldase and its cognate lipases from Acinetobacter sp. in order to unveil the foldingmechanism of lipases.The bacterial strain Acinetobacter sp. XMZ-26(ACCC05422) was isolated from soil samplesobtained from glaciers in Xinjiang Province, China. A novel operon containing a lipase gene (lip26) andits specific foldase gene (lif26) was isolated by creating and screening the genomic library and thenusing genomic walking from Acinetobacter sp. XMZ-26. The lip26gene was located in the downstreamof the lif26gene (the accession numbers in GenBank database: GQ227699and GQ227701). The aminoacid sequence of Lip26/Lif26showed only46.4and37.3%identity with the LipA/LipB (Lif) sequencesfrom Acinetobacter sp. SY-01. The expressed recombinant Lip26formed inactive inclusion bodies in E.coli. However, the active Lip26was refolded by the dilution refolding method with the assistance ofpurified recombinant Lif26, and the refolded Lip26had a high specific activity. Lip26hydrolyzedp-nitrophenyl (pNP) esters of fatty acids with C2to C16acyl chain lengths and had a substratepreference for pNP myristate. Maximal Lip26activity was dependent on both the temperature (55°C)and pH (9.0). In addition, Lip26was capable of maintaining its activity in the presence of manydetergents and organic solutions, and its activity was enhanced by the presence of Ca2+, Mn2+, and Ba2+.To directly obtain active Lip26, the E. coli strain was also co-transformed with two expression plasmidscontaining the lip26and lif26genes. The co-expression of both proteins in vivo resulted in theexpression of half of the recombinant Lip26as a soluble protein with demonstrable lipase activity. As asteric chaperone, the lipase specific foldase Lif26was required for the correct folding of the lipaseLip26.Lif26was shown to interact with Lip26via the yeast two hybrid assay in vivo, and the associationwas confirmed in vitro by GST pull-down experiments. Moreover, the protein interaction between theLif and lipase was specifically detected by the yeast two hybrid assay of the Lifs and lipases fromdifferent stains. In order to unveil the protein interation between Lif26and Lip26, the three dimensionalstructure of the Lif26-Lip26complex was homology modeled using the Lif-LipA complex from Burkholderia glumae (PDB ID:2ES4) as the template. It revealed that Lif26consisted of elevenα-helixes, with the N-terminal minidomain (α1-α3) and C-terminal minidomain (α9-α11) engulfing thelipase Lip26. To identify the key regions of Lif26involving in the interaction with Lip26, Lif26/Lif4chimaera and deleted mutants were constructed. The results showed that the α11in the C-terminalminidomain of Lif26was essential for its association with Lip26, whereas the α1-helix in theN-terminus was not.In order to study the molecular determinants for the interaction between the Lif26and Lip26, asystematic strategy was used. It included a homology model-based screening of residues, moleculardynamics (MD) simulation-based calculating interaction energy, and followed by site-directedmutagenesis to alter individual screened residues. One conserved amino acid, Arg332, in the C-terminalmini-domain of Lif26was identified as an important residue involving in the protein interaction withthe Lip26. The residue Arg332could not replaced by the any other residues detected by the saturatesite-directed mutagenesis, and it formed a conserved and stable salt bridge with the conserved Glu112of Lip26, which may contribute to the specificity of binding. Besides, the residues surrounding Arg332such as Trp288in the α9probably contributed to stabilize the Arg332residue in a proper conformationfor interaction with the Lip26.