Molecular characterization of enterobacterial pldA genes encoding outer membrane phospholipase A

Abstract

The pid4 gene of Escherichia coli encodes an outer membrane phospholipase A. A strain carrying the most commonly used mutant pkld allele appeared to express a correctly assembled PldA protein in the outer membrane. Nucleotide sequence analysis revealed that the only difference between the wild type and the mutant is the replacement of the serine residue in position 152 by phenylalanine. Since mutants that lack thepld4 gene were normally viable under laboratory conditions and had no apparent phenotype except for the lack of outer membrane phospholipase activity, the exact role of the enzyme remains unknown. Nevertheless, the enzyme seems to be important for the bacteria, since Western blotting (immunoblotting) and enzyme assays showed that it is widely spread among species of the family Enterobacteriaceae. To characterize the PldA protein further, the pid4 genes of Salmonella typhimurium, Klebsiella pneumoniae, and Proteus vulgaris were cloned and sequenced. The cloned genes were expressed in E. coli, and their gene products were enzymatically active. Comparison of the predicted PldA primary structures with that of E. coli PldA revealed a high degree of homology, with 79%o of the amino acid residues being identical in all four proteins. Implications of the sequence comparison for the structure and the structure-function relationship of PldA protein are discussed.Most bacterial outer membrane proteins are involved in the transport of nutrients across this membrane by forming pores or receptors. In addition, these membranes contain a few enzymes, e.g., the detergent-resistant outer membrane phospholipase A or PldA protein of Escherichia coli. Several activities reside in this enzyme, i.e., those of phospholipases Al and A2 and of 1-acyl and 2-acyl lysophospholipase and lipase, with the phospholipase A1 activity being six times greater than the phospholipase A2 activity (24). The PIdA protein is encoded by the pldA gene, the nucleotide sequence of which has been determined (23). This gene codes for a 30-kDa mature protein of 269 amino acid residues preceded by a signal sequence of 20 amino acid residues. The three-dimensional structure of the enzyme is unknown. Like other outer membrane proteins, PldA protein lacks hydrophobic sequences long enough to span the lipid bilayer. Therefore, its structure might be comparable with those of the porins which have recently been determined (10,54,55). In these outer membrane proteins, the polypeptide chain traverses the outer membrane repeatedly as antiparallel 3-strands. A comparison of the outer membrane protein PhoE among three species of the family Enterobacteriaceae has revealed that during evolution some parts of the polypeptide have undergone more extensive divergence than others (49). For PhoE, these variable regions correspond to cell surface-exposed segments. A similar finding was reported for the OmpA protein (7). Thus, sequence comparisons can be helpful in predicting the topology of outer membrane proteins.The exact function of the PldA protein is unknown. The pro...

Bacterial phospholipase A: structure and function of an integral membrane phospholipase

“…The rather hydrophilic character and the lack of long hydrophobic amino acid stretches, indicated that the interaction of OMPLA with the membrane di¡ered from integral membrane proteins such as bacteriorhodopsin [7]. Circular dichroism (CD) measurements indicated a high L-strand content [26] and this led Brok et al [27] to propose a molecular model with a L-barrel architecture. Electron crystallography con¢rmed the L-barrel character; projection maps showed an oval ring-like density large enough to accommodate 12 L-strands.…”

“…This suggested that a serine might be a catalytic residue. Later, the ¢nding of an inactive Ser152Phe variant of OMPLA suggested that residue 152 was the active site serine [27]. This hypothesis was rejected, however, when site directed mutagenesis demonstrated some tolerance at this position (Ser152Asn and Ser152Thr) [32].…”

“…However, the asparagine residue shows only a modest contribution to catalysis, as an Asn156Ala mutant retained 5% residual activity [46]. Moreover, while Ser144 and His142 are strictly conserved, Asn156 can vary among the di¡erent species; an aspartate residue is observed in Panthoea agglomerans OM-PLA [27] and a glutamine residue is observed in H.…”

Crystallization and preliminary X‐ray analysis of outer membrane phospholipase A fromEscherichia coli

“…It is obvious that the E. coli OMPLA and all other membrane phosphotipases, embedded in their own substrate, require a very strict regulatory mechanism for their enzymatic activity to prevent unwanted lysis of the cell envelope. In normally growing cells OMPLA seems to be dormant and E. coli mutants missing the pldA gene are normally viable under laboratory conditions, which suggests that the protein has no essential role [6]. Nevertheless, OMPLA might be important for growth of the bacteria *Corresponding author.…”

“…in their natural environment since it occurs widespread and is well-conserved among Enterobacteriaceae, including pathogenic species [6].…”