Studies on the characteristics and secretory mechanism of PalA, pseudomonas sp. lipolytic enzyme

Abstract

A gene cloned from Pseudomonas sp., called HSM0414 directed the production of lipolytic activity. From the sequencing data, an open reading frame encoding a protein of 636 amino acids with an estimated molecular mass of 68.9 kDa was found. The product of the gene, named PalA, was processed by removal of an N-terminal signal peptide to yield a 66.4 kDa mature protein. The deduced amino acid sequence of PalA did not contain the typical GXSXG motif found in most esterases and lipases, suggesting that the protein is a member of a novel GDSL family of lipolytic enzymes. The predicted 636 amino acids in the protein encoded by palA showed 66% and 58% identity with P. aeruginosa PAO1 EstA and putative ORF of P. putida trpE-TrpG region, respectively. Amino acid sequence alignments led to the prediction that this lipolytic enzyme was an autotransporter protein that possessed a C-terminal β-barrel domain, allowing the secretion of the N-terminal catalytic domain harbouring the lipolytic activity. PalA is composed of two parts, the N-terminal region (Ala1-Ala296) similar to the GDSL lipases and C-terminal region (Leu320-Phe612) to the autotransporter. The expression of PalA in E. coli, subsequent cell fractionation, whole-cell ELISA, external protease treatment, and in vitro refolding study revealed that the enzyme was associated with the cellular outer membranes. Autotransporter mode of surface presentation in Gram-negative bacteria requires a hypothetical C-terminal β-barrel which makes up an aqueous channel in the outer membrane. In this report, we provide biochemical and structural evidence demonstrating that the pore size of the β-barrel conduit is important to deliver the N-domain to cell surface. Among the all autotransporter domains, two strictly conserved residues $(Pro^{478} and Gly^{576} in PalA)$ are converted to other various residues using site-directed mutagenesis. This investigation was made into the different pore-size mutants, affecting the active foldin...