The importance of glycine-30 for enzymatic activity of phospholipase A2

Bekkers, A.C.A.P.A.; Franken, P.; Toxopeus, Ellis; Verheij, Hubertus M.; Haas, G.H. de

doi:10.1016/0167-4838(91)90479-j

Cited by 24 publications

(10 citation statements)

References 21 publications

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“…More specifically, we find that two conserved glycines belonging to the consensus segment X 1 CG 1 XG 2 and involved in metal binding are replaced by other small amino acids (S,T,V,A) in the TbSP1‐PLA 2 and in its relatives. Since the substitution of the G 1 residue with a serine is known to cause a 10‐ to 20‐fold reduction in catalytic activity (Bekkers et al ., 1991), it is unlikely that the structural context of the Ca 2+ coordination site of TbSP1 is the same as that of the other PLA 2 groups. It is interesting to note, in this regard, the presence in the Ca 2+ ‐binding region of TbSP1‐PLA 2 s of three conserved aspartate residues (Figure 2).…”

Section: Discussionmentioning

confidence: 99%

A nutrient-regulated, dual localization phospholipase A2 in the symbiotic fungus Tuber borchii

Soragni

Bolchi

Balestrini

et al. 2001

EMBO J

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Important morphogenetic transitions in fungi are triggered by starvation-induced changes in the expression of structural surface proteins. Here, we report that nutrient deprivation causes a strong and reversible up-regulation of TbSP1, a surface-associated, Ca 2+ -dependent phospholipase from the mycorrhizal fungus Tuber borchii. TbSP1 is the ®rst phospholipase A 2 to be described in fungi and identi®es a novel class of phospholipid-hydrolyzing enzymes. The TbSP1 phospholipase, which is synthesized initially as a preprotein, is processed ef®ciently and secreted during the mycelial phase. The mature protein, however, also localizes to the inner cell wall layer, close to the plasma membrane, in both free-living and symbiosisengaged hyphae. It thus appears that a dual localization phospholipase A 2 is involved in the adaptation of a symbiotic fungus to conditions of persistent nutritional limitation. Moreover, the fact that TbSP1-related sequences are present in Streptomyces and Neurospora, and not in wholly sequenced non-®lamen-tous microorganisms, points to a general role for TbSP1 phospholipases A 2 in the organization of multicellular ®lamentous structures in bacteria and fungi.

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Section: Discussionmentioning

confidence: 99%

A nutrient-regulated, dual localization phospholipase A2 in the symbiotic fungus Tuber borchii

Soragni

Bolchi

Balestrini

et al. 2001

EMBO J

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“…Calcium ions are an absolute requirement for phospholipase A activity (2,16). Indeed, the activity of the C. coli PldA showed an absolute requirement for this cation.…”

Section: Discussionmentioning

confidence: 99%

Molecular characterization of pldA, the structural gene for a phospholipase A from Campylobacter coli, and its contribution to cell-associated hemolysis

et al. 1997

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A gene (pldA) encoding a 35.0-kDa protein with significant homology to the Escherichia coli outer membrane phospholipase was identified upstream of an operon encoding an enterochelin transport system in Campylobacter coli. The results of this study suggest that this gene encodes an outer membrane phospholipase A in C. coli. First, expression of the pldA gene product in a PldA-deficient mutant of E. coli led to the restoration of phospholipase A activity. The recombinant product also partitioned to the outer membrane, suggesting that it may be similarly located in C. coli. Second, heterologous overexpression in E. coli, followed by in vitro folding and purification of C. coli PldA, resulted in pure protein which displayed calcium-dependent lysophospholipase and phospholipase A activities in vitro. Finally, mutants of C. coli in which the pldA gene had been inactivated by allelic exchange were deficient in phospholipase A activity. Phospholipases are associated with lysis of erythrocytes by a number of bacterial pathogens. The pldA mutant was shown to have a reduced hemolytic activity compared to the wild-type strain, suggesting a role for the phospholipase A in the lysis of erythrocytes by C. coli. Since hemolysins are intimately associated with the disease-causing potential of a number of bacterial pathogens, it is likely that the phospholipase A plays some role in Campylobacter virulence. The thermophilic Campylobacter species, particularly Campylobacter jejuni and Campylobacter coli, are established as the most frequently isolated bacteria that cause acute diarrheal disease in humans (11, 27). In view of the incidence of Campylobacter infection, however, there still remains a general absence of information about the mechanisms involved in the pathogenesis of infection (36). Once ingested, the bacteria are thought to colonize the intestinal mucosa, with both flagella and their corkscrew shape enabling them to penetrate this viscous environment. The importance of flagella in colonization of the mucosa has been demonstrated in studies using aflagellate mutants (6, 43). Campylobacters are believed to adhere to and invade intestinal epithelial cells, and adhesion to cell lines has been demonstrated in vitro (18, 41). Although C. jejuni and C. coli have been reported to produce a variety of toxins (12, 24, 30), definitive reports have not yet appeared and the involvement of these proteins in the pathogenesis of infection remains controversial (13, 28). Hemolysins are intimately associated with the virulence of a number of bacterial pathogens (20), and there is increasing evidence that campylobacters may produce one or more hemolysins (17, 23, 29). The modes of action of hemolysins are diverse, but there are several studies which correlate the ability of bacteria to lyse erythrocytes with phospholipase activity (1, 25). These enzymes cleave membrane phospholipids into lysophospholipids, lysophosphatides, and free fatty acids, leading to membrane lysis. In this study, we report the genetic characterization of a gene (p...

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“…In this context, the requirement of a calcium ion is considered to be generally essential for the activity of PLA 2 15–24. The calcium ion is known to bind tightly to the protein molecule in the so‐called cage‐like calcium binding loop25, 26 and supports a highly ordered conformation of one of the walls of the hydrophobic channel, which in turn facilitates the passage of the substrate to the active site. In spite of the well established role of calcium ion, the various aspects related to its concentration and PLA 2 activity have not yet been clearly defined.…”

Section: Introductionmentioning

confidence: 99%

Crystal structure of a calcium‐induced dimer of two isoforms of cobra phospholipase A₂ at 1.6 Å resolution

et al. 2005

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The calcium-induced formation of a complex between two isoforms of cobra venom phospholipase A2 reveals a novel interplay between the monomer-dimer and activity-inactivity transitions. The monodispersed isoforms lack activity in the absence of calcium ions while both molecules gain activity in the presence of calcium ions. At concentrations higher than 10 mg/ml, in the presence of calcium ions, they dimerize and lose activity again. The present study reports the crystal structure of a calcium-induced dimer between two isoforms of cobra phospholipase A2. In the complex, one molecule contains a calcium ion in the calcium binding loop while the second molecule does not possess an intramolecular calcium ion. However, there are two calcium ions per dimer in the structure. The second calcium ion is present at an intermolecular site and that is presumably responsible for the dimerization. The calcium binding loops of the two molecules adopt strikingly different conformations. The so-called calcium binding loop in the calcium-containing molecule adopts a normal conformation as generally observed in other calcium containing phospholipase A(2) enzymes while the conformation of the corresponding loop in the calcium free monomer deviates considerably with the formation of a unique intraloop Gly33 (N)-Cys27 (O) = 2.74 A backbone hydrogen bond. The interactions of Arg31 (B) with Asp49 (A) and absence of calcium ion are responsible for the loss of catalytic activity in molecule A while interactions of Arg2 (B) with Tyr52 (B) inactivate molecule B.

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The importance of glycine-30 for enzymatic activity of phospholipase A2

Cited by 24 publications

References 21 publications

A nutrient-regulated, dual localization phospholipase A2 in the symbiotic fungus Tuber borchii

A nutrient-regulated, dual localization phospholipase A2 in the symbiotic fungus Tuber borchii

Molecular characterization of pldA, the structural gene for a phospholipase A from Campylobacter coli, and its contribution to cell-associated hemolysis

Crystal structure of a calcium‐induced dimer of two isoforms of cobra phospholipase A₂ at 1.6 Å resolution

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The importance of glycine-30 for enzymatic activity of phospholipase A2

Cited by 24 publications

References 21 publications

A nutrient-regulated, dual localization phospholipase A2 in the symbiotic fungus Tuber borchii

A nutrient-regulated, dual localization phospholipase A2 in the symbiotic fungus Tuber borchii

Molecular characterization of pldA, the structural gene for a phospholipase A from Campylobacter coli, and its contribution to cell-associated hemolysis

Crystal structure of a calcium‐induced dimer of two isoforms of cobra phospholipase A2 at 1.6 Å resolution

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Crystal structure of a calcium‐induced dimer of two isoforms of cobra phospholipase A₂ at 1.6 Å resolution