Dataset Information

Atomic resolution (0.97 A) structure of the triple mutant (K53,56,121M) of bovine pancreatic phospholipase A2.

ABSTRACT: The enzyme phospholipase A2 catalyzes the hydrolysis of the sn-2 acyl chain of phospholipids, forming fatty acids and lysophospholipids. The crystal structure of a triple mutant (K53,56,121M) of bovine pancreatic phospholipase A2 in which the lysine residues at positions 53, 56 and 121 are replaced recombinantly by methionines has been determined at atomic resolution (0.97 A). The crystal is monoclinic (space group P2), with unit-cell parameters a = 36.934, b = 23.863, c = 65.931 A, beta = 101.47 degrees. The structure was solved by molecular replacement and has been refined to a final R factor of 10.6% (Rfree = 13.4%) using 63,926 unique reflections. The final protein model consists of 123 amino-acid residues, two calcium ions, one chloride ion, 243 water molecules and six 2-methyl-2,4-pentanediol molecules. The surface-loop residues 60-70 are ordered and have clear electron density.

SUBMITTER: Sekar K

PROVIDER: S-EPMC1952368 | biostudies-literature | 2005 Jan

REPOSITORIES: biostudies-literature

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Atomic resolution (0.97 A) structure of the triple mutant (K53,56,121M) of bovine pancreatic phospholipase A2.

Sekar K K Rajakannan V V Gayathri D D Velmurugan D D Poi M-J MJ Dauter M M Dauter Z Z Tsai M-D MD

Acta crystallographica. Section F, Structural biology and crystallization communications 20040925 Pt 1

The enzyme phospholipase A2 catalyzes the hydrolysis of the sn-2 acyl chain of phospholipids, forming fatty acids and lysophospholipids. The crystal structure of a triple mutant (K53,56,121M) of bovine pancreatic phospholipase A2 in which the lysine residues at positions 53, 56 and 121 are replaced recombinantly by methionines has been determined at atomic resolution (0.97 A). The crystal is monoclinic (space group P2), with unit-cell parameters a = 36.934, b = 23.863, c = 65.931 A, beta = 101.4 ...[more]

PMID: 16508077

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Project description:Recent findings have indicated that secreted phospholipases A2 (sPLA2s) have anti-inflammatory functions, including relief of symptoms in a mouse model of mastitis. This prompted us to investigate the therapeutic application of sPLA2, PLA2G1B, for bovine mastitis. Initial testing of PLA2G1B's effect on bovine mammary epithelial cell (bMEC) line PS revealed no changes in cell viability or cytokine-secretion pattern. However, when cells were first treated with lipopolysaccharide endotoxin (LPS) or live bacteria (Escherichia coli or Staphylococcus aureus), incubation with PLA2G1B significantly improved cell viability, suggesting involvement of sPLA2s in protecting membranes from lipid-peroxidation damage, rather than a bactericidal action. When PLA2G1B was applied simultaneously with LPS, a significant short-term reduction in interleukin-8 secretion was observed compared with bMECs treated only with LPS, supporting previous reports that PLA2G1B affects interleukin-8 signaling in similar cells. Following the favorable outcome of the in vitro experiments, we tested PLA2G1B in vivo by mammary infusion into infected glands. In one of a small sample (n = 4) of lactating cows chronically infected with Streptococcus dysgalactiae, a single PLA2G1B treatment completely cleared inflammation and bacteria, demonstrating its potential to cure subclinical mastitis. PLA2G1B treatment did not affect coagulase-negative staphylococci infection. These types of mastitis may involve formation of a resistant biofilm, and its elimination may relate to sPLA2s' characteristic ability to aggregate with cellular debris, facilitating their internalization by macrophages. In a bovine model of clinical mastitis based on introduction of E. coli via the streak canal, a single mammary infusion of PLA2G1B led to faster recovery to pre-infection milk-yield levels and decrease of somatic cell counts. In this case, all of sPLA2s' modes of resolving inflammation may apply, including competitive binding of the sPLA2s' receptor, the inactivation of which confers resistance to endotoxic shock. Hence, this study strongly supports further research into PLA2G1B as a cure for bovine mastitis.

Dataset Information

Atomic resolution (0.97 A) structure of the triple mutant (K53,56,121M) of bovine pancreatic phospholipase A2.

Publications

Atomic resolution (0.97 A) structure of the triple mutant (K53,56,121M) of bovine pancreatic phospholipase A2.

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