<i>Escherichia coli</i>Outer Membrane Phospholipase A:  Role of Two Serines in Enzymatic Activity

Brok, Ronald G. P. M.; Belandia, I U; Dekker, Niek; Tommassen, Jan; Verheij, Hubertus M.

doi:10.1021/bi952970i

Cited by 25 publications

(30 citation statements)

References 46 publications

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“…This is the more interesting since it has been suggested that the catalytic center of the enzyme contains a classical Asp-His-Ser triad (see reference 24 and references therein). The involvement of Ser-144 as the active-site serine residue, as originally determined by chemical modification (24), was recently confirmed by Brok et al (10) in a site-directed mutagenesis study. Furthermore, the involvement of His-142 in the catalytic mechanism has been demonstrated (9).…”

Section: Discussionmentioning

confidence: 69%

“…An alternative explanation for our results is that loop L4 has a dual location with respect to the membrane plane, being partly exposed and partly on the periplasmic side. It is important in this respect to note that loop L4 contains the active-site serine, Ser-144, and two other residues, His-142 and Ser-152, which are required for enzymatic activity (8)(9)(10). Thus, one could envisage a model for the activation of the enzyme that involves a movement of L4 relative to the membrane through the ␤-barrel.…”

Section: Discussionmentioning

confidence: 99%

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Topology of the outer membrane phospholipase A of Salmonella typhimurium

et al. 1997

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The outer membrane phospholipase A (OMPLA) of Enterobacteriaceae has been proposed to span the membrane 14 times as antiparallel amphipathic ␤-strands, thereby exposing seven loops to the cell surface. We have employed the epitope insertion method to probe the topology of OMPLA of Salmonella typhimurium. First, missense mutations were introduced at various positions in the pldA gene, encoding OMPLA, to create unique BamHI sites. These BamHI sites were subsequently used to insert linkers, encoding a 16-amino-acid B-cell epitope. Proper assembly of all mutant proteins was revealed by their heat modifiability in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The accessibility of the inserted epitopes was assessed. Immunofluorescence analysis of intact cells with antibodies against the inserted epitope showed that three of seven predicted loops are indeed cell surface exposed. Trypsin accessibility experiments verified the cell surface exposure of two additional loops and provided support for the proposed periplasmic localization of three predicted turns. For two other predicted exposed loops, the results were not conclusive. These results support to a large extent the proposed topology model of OMPLA. Furthermore, the observation that the substitutions Glu66Pro and Glu247Gly virtually abolished enzymatic activity indicates that these residues might play a major role in catalysis.Most bacterial outer membrane proteins are involved in transport processes, either as pore-forming proteins or as receptors. The outer membrane phospholipase A (OMPLA) (also designated PldA protein, after its structural gene pldA) is one of the few enzymes present in this membrane. This Ca 2ϩ -dependent enzyme has hydrolytic activity towards phospholipids and lipids (23,31,37). The pldA gene is widely distributed among members of the family Enterobacteriaceae, and the primary structure of this protein is highly conserved (8). The pldA genes of Escherichia coli and Salmonella typhimurium encode 30-kDa proteins of 269 amino acid residues, preceded by signal sequences of 20 residues (8,22). The function of OMPLA is unknown. It has been shown that E. coli OMPLA is required for efficient secretion of bacteriocins (28, 32), but it is unlikely that this is the primary function of the protein. Since OMPLA is an outer membrane protein, it is embedded in its substrate. However, enzymatic activity is detected only when the integrity of the outer membrane is affected, e.g., by heat shock (18) or by phage-induced lysis (16). The mechanism of enzymatic activation of OMPLA, however, has not yet been elucidated. The structures and functions of many water-soluble phospholipases are known in detail, but OMPLA has no sequence homology with any of these well-characterized phospholipases.To understand the catalytic and activation mechanisms of this intriguing enzyme, knowledge of its structure is needed. An E. coli OMPLA derivative has been overproduced, purified, and refolded with good yields (19). Although the refolded protein did crystallize (5),...

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Section: Discussionmentioning

confidence: 69%

Section: Discussionmentioning

confidence: 99%

Topology of the outer membrane phospholipase A of Salmonella typhimurium

et al. 1997

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“…The active site contains Ser 144 and His 142, as established from chemical modification 12 and site-directed mutagenesis experiments 13,14 , and is located on the exterior of the ␤-barrel, positioned just outside the outer leaflet ring of aromatic residues. Besides the serine and histidine, the active site contains an asparagine residue (Asn 156).…”

mentioning

confidence: 99%

Structural evidence for dimerization-regulated activation of an integral membrane phospholipase

Snijder

Ubarretxena-Belandia

Blaauw

et al. 1999

Nature

195

231

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“…1%). This has been observed more often for serine-cysteine mutations in the serine hydrolase family [32].…”

Section: Active Sitementioning

confidence: 83%

“…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]. De¢nite evidence for a serine as catalytic nucleophile came from inhibition studies with hexadecanesulphonyl £uoride.…”

Section: Active Sitementioning

confidence: 99%