Functional importance of calcium binding sites in outer membrane phospholipase A.
Outer membrane phospholipase A (OMPLA) is an integral membrane enzyme that hydrolyses phospholipids requiring Ca(2+) as cofactor. In vitro studies have shown that OMPLA is only active as a dimer. The structures of monomeric and dimeric OMPLA provided possible clues to the activation process. In the inhibited dimeric species calcium ions are located at the dimer interface ideally suited to stabilise the oxyanion intermediates formed during catalysis. The side chain hydroxyl function of Ser152 is one of the ligands of this interfacial calcium. In the crystal structure of monomeric OMPLA the interfacial calcium site is lacking, but calcium was found to bind at a site involving the carboxylates of Asp149 and Asp184. In the current study the relevance of the identified calcium sites has been studied by site-directed mutagenesis. The Ser152Asn variant confirmed the importance of the interfacial calcium site for catalysis, and also demonstrated that this site is essentially involved in the dimerisation process. Replacements of the ligands in monomeric OMPLA, i.e. Asp149Asn, Asp149Ala and Asp184Asn, only showed minor effects on catalytic activity and dimerisation. A stronger effect observed for the variant Asp184Ala was explained by the proximity of Asp184 to the catalytically important Ser152 residue. We propose that Asp149 and Asp184 provide an electronegative funnel that may facilitate Ca(2+) transfer to the interfacial calcium site.
Kingma
Roelie L
RL
Department of Membrane Enzymology, Centre for Biomembranes and Lipid Enzymology, Institute of Biomembranes, Utrecht University, P.O. Box 80054, 3508 TB Utrecht, Netherlands.
Snijder
Harm J
HJ
Dijkstra
Bauke W
BW
Dekker
Niek
N
Egmond
Maarten R
MR
D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry / Q000378:metabolism*; D002850:Chromatography, Gel; D019281:Dimerization; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010741:Phospholipases A / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D054466:Phospholipases A1
D001425:Bacterial Outer Membrane Proteins; D010741:Phospholipases A; D054466:Phospholipases A1; C412876:outer membrane phospholipase A; D002118:Calcium