Structures of the catalytic site mutants D99A and H48Q and the calcium-loop mutant D49E of phospholipase A₂

Abstract

Crystal structures of the active-site mutants D99A and H48Q and the calcium-loop mutant D49E of bovine phospholipase A 2 have been determined at around 1.9 A Ê resolution. The D99A mutant is isomorphous to the orthorhombic recombinant enzyme, space group P2 1 2 1 2 1 . The H48Q and the calciumloop mutant D49E are isomorphous to the trigonal recombinant enzyme, space group P3 1 21. The two active-site mutants show no major structural perturbations. The structural water is absent in D99A and, therefore, the hydrogen-bonding scheme is changed. In H48Q, the catalytic water is present and hydrogen bonded to Gln48 N, but the second water found in native His48 is absent. In the calcium-loop mutant D49E, the two water molecules forming the pentagonal bipyramid around calcium are absent and only one O atom of the Glu49 carboxylate group is coordinated to calcium, resulting in only four ligands.

Active-site mutagenesis of a Lys49-phospholipase A2: biological and membrane-disrupting activities in the absence of catalysis

“…onstrate that the secondary, tertiary and quaternary structures of the native, recombinant wild-type and mutant rBthTx-I proteins are indistinguishable. This conclusion is in agreement with previous site-directed mutagenesis results in bovine pancreatic class II Asp%*-PLA # , which also demonstrated that mutations in the active-site region do not perturb significantly the global structure of the protein [16,41].…”

Lysis of Human Immunodeficiency Virus Type 1 by a Specific Secreted Human Phospholipase A2

“…Virus preparations were incubated with purified sPLA 2 -X or a different catalytically inactive mutant, ⌬3sPLA 2 -X (H46N, D47E, and Y50F mutations) (9,19), prior to infection of the human T-cell leukemia cell line A3R5, a subline of A3.01 cells (10) expressing both CCR5 and CXCR4. Flow cytometric analysis of intracellular Gag protein was used to assess viral replication.…”

Crystal Structures of the Free and Anisic Acid Bound Triple Mutant of Phospholipase A2

“…In most of the bovine pancreatic PLA2 and its mutant structures studied so far, 5,22,27,33,34 the surface loop residues were always found to be disordered, except in the high-resolution (1.5 Å ) orthorhombic form 26 of the recombinant PLA2. Again in the present free triple mutant structure, the electron density is not clear and the surface loop residues are disordered.…”

“…The surface loop residues (60 -70) and the calcium-binding loop(27)(28)(29)(30)(31)(32)(33)(34) are indicated with the residue numbers at the beginning as well as at the ends. This Figure was produced using the program MOLSCRIPT 44.…”