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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...

Es:sr.herichia coli outer-membrane phospholipase A (OMPLA) is thought to be a tnember of the class of serine hydrolases, having a classical Asp-His-Ser catalytic triad [Horrevoets, A. J. G., Verheij, k1. M. & de Haas, G. H. (199'1) Eicr: .I. Biocherrr. 198, 247-2531. To idenlify the histidinc residue that is important for catalytic activity, the four histidine residues in E. c d i OMPLA that arc conserved in other enterobacterial OMPLA enzymes werc replaced by cysteine residues using PCR-directed, site-specific mutagenesis. The resulting mutant proteins were all well cxpressed and displayed heat modifiability, indicating that they were properly folded. Enzyme assays showed that only the Hisl42Cys mutant protein was lacking enzymatic activity. In addition, a His142Gly mutant protein appeared to he inactive. These result? show that His142 is important for the enzymatic activity of OMPLA.Keqrwords: outer-membrane protein; phospholipase A; active site; site-directed mutagenesis; Escherichiu coli.Outer-membrane phospholipase A (OMPLA) of Es:Jheric.hiti coli is a 30-kDa protein that is eiicodcd by the p1dA gene. It is widely disseminated among members of the Enterobacteriaceae family (Brok et al., 1994), but its physiological role is still unknown. It has been shown that R. coli OMPLA is required for efficient secretion of bacteriocins (Pugsley and Schwiutn, 1984; Luirink et al., 1986), but it is unlikely that this is the primary [unction of the protein. OMPLA activity must be well regulated since otherwise the enzyme would degrade the cell envelope. In normally growing cells, OMPLA appears to be dormant (A~idet et al., 1974). However, high OMPLA activity can be induced by damaging the membrane, e.g. by phage-induced lysis (Cronan and Wulff, 1960) or by temperalure shock (de Geus et al., 19x3). The 1~ldA genes of E. coli (Homtnii et al., 1984), Snlm!pplonell~i typhimurium, Klebsiellci pneurnoniuf,, and Proteus vulgnris (Rrok et al., 1094) have been cloned and sequenced, and they show high mutual sequence similarity.OMPLA has several enzymatic activities, i.e. those of phospholipases A, and A,, 1 -acyl-and 2-acyl-lysophospholipase, and diacylglycerol lipase, with (he phospholipase A, activity being six times higher thnn the phospholipase A, activity (Horrevoets et nl., 1989). It has bcen suggested (Horrevoets et al., 1991) that OMPLA belongs 10 thc class of serine hydrolases whose members catalyse cster hydrolysis via an ucyl cnzyme interniediate. However, in contrast to serine proteases (Kraut, 1977), OMPI>A is not inactivated by water-solublc serine hydrolase inhibitors like diisopropyltluorophosphate and phcnylmethylsulfotiyl fluoride. Only water-insoluble inhibitors, like n-hexadecylsulfonyl C~f~rrt..s~~o~~rluncr to H. M. Verheij,

In the outer membrane phospholipase A (OMPLA) of Escherichia coli, Ser144 has previously been identified by chemical modification as the active site serine residue. In a specific OMPLA-negative mutant strain, the pldA gene coding for OMPLA was shown to differ from the wild-type gene by a single point mutation, resulting in the substitution of Ser152 by phenylalanine. The role in catalysis of these two serine residues in OMPLA was investigated by site-directed mutagenesis. Ser144 and Ser152 were replaced one at the time by either alanine, valine, phenylalanine, threonine, or cysteine. Ser152 was furthermore replaced by asparagine. Replacement of Ser144 by cysteine resulted in 1% residual activity, whereas the other substitutions at this position yielded virtually inactive enzymes. Substitution of Ser 152 by threonine or asparagine resulted in 40% and 2% residual activity respectively, whereas all other substitutions at this position resulted in the loss of enzymatic activity. We propose that Ser144 is the nucleophile in catalysis, and that Ser152 is involved in hydrogen bonding either to the catalytic triad or in the oxyanion hole.

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