PDBsum entry 2phi

Hydrolase (carboxylic ester)

PDB id: 2phi

Contents

Protein chains

124 a.a. *

Metals

_CA ×3

Waters ×195

* Residue conservation analysis

PDB id:

2phi

Name:

Hydrolase (carboxylic ester)

Title:

A large conformational change is found in the crystal structure of the porcine pancreatic phospholipase a2 point mutant f63v

Structure:

Phospholipase a2. Chain: a, b. Engineered: yes

Source:

Sus scrofa. Pig. Organism_taxid: 9823. Organ: pancreas

Biol. unit:

Dimer (from PQS)

Resolution:

2.20Å R-factor: 0.176

Authors:

B.W.Dijkstra,M.M.G.M.Thunnissen,K.H.Kalk,J.Drenth

Key ref:

M.M.Thunnissen et al. (1993). Crystal structure of a porcine pancreatic phospholipase A2 mutant. A large conformational change caused by the F63V point mutation. J Mol Biol, 232, 839-855. PubMed id: 8355274

Date:

08-Apr-93 Release date: 15-Jul-93

Protein chains

P00592  (PA21B_PIG) -  Phospholipase A2, major isoenzyme from Sus scrofa

Seq: 146 a.a.

Struc: 124 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class: E.C.3.1.1.4 - phospholipase A2.
• Reaction: a 1,2-diacyl-sn-glycero-3-phosphocholine + H2O = a 1-acyl-sn-glycero-3- phosphocholine + a fatty acid + H⁺
• Cofactor: Ca(2+)

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

reference

J Mol Biol 232:839-855 (1993)

PubMed id: 8355274

Crystal structure of a porcine pancreatic phospholipase A2 mutant. A large conformational change caused by the F63V point mutation.

M.M.Thunnissen, P.A.Franken, G.H.de Haas, J.Drenth, K.H.Kalk, H.M.Verheij, B.W.Dijkstra.

ABSTRACT

The highly homologous bovine and porcine pancreatic phospholipase A2 (85% amino acid residue identity) show a large conformational difference in the loop from residue 59 to 71. In bovine phospholipase A2 residues 59 to 66 adopt an alpha-helix conformation, while residues 67 to 71 are in a surface loop. Residues 59 to 66 in the porcine enzyme have a random coil conformation, and residues 67 to 71 form a short 3(10)-helix. It has been suggested that most probably this conformational difference is caused by the substitution Val63 (bovine) to Phe63 (porcine) in the otherwise invariant loop 59 to 70. To test this hypothesis, a mutant porcine phospholipase A2 was constructed in which residue Phe63 was replaced by a Val. The activity of this F63V mutant towards aggregated substrates was about half the activity of wild-type porcine phospholipase A2, but significantly different from that of the bovine enzyme. The affinity for zwitterionic interfaces was found to be intermediate between porcine and bovine phospholipase. The mutation did not have any effect on the stability of the enzyme towards denaturation by guanidine.HCl. The F63V mutant was crystallized in space group P2(1)2(1)2(1) with cell dimensions a = 79.88 A, b = 65.23 A, c = 52.62 A, with two molecules per asymmetric unit. Its three-dimensional structure was solved by molecular replacement methods, and refined to a crystallographic R-factor of 17.6% for all data between 10 and 2.2 A resolution. In one molecule the 58 to 71 loop is in very weak density, suggesting a high degree of disorder or flexibility. The conformation of the same loop in the other molecule could be determined unambiguously. It shows a conformation which resembles more that of bovine phospholipase A2 than that of porcine phospholipase. It is concluded that indeed the single F63V substitution causes a dramatic conformational change.