Dimerization Regulates the Enzymatic Activity of Escherichia coli Outer Membrane Phospholipase A

Abstract

The outer membrane phospholipase A (OMPLA) of Escherichia coli is present in a dormant state in the cell envelope. The enzyme is activated by various processes, which have in common that they perturb the outer membrane. Kinetic experiments, chemical cross-linking, and analytical ultracentrifugation were carried out with purified, detergent-solubilized OMPLA to understand the underlying mechanism that results in activation. Under conditions in which the enzyme displayed full activity, OMPLA was dimeric. High detergent concentrations or very dilute protein concentrations resulted in low specific activity of the enzyme, and under those conditions the enzyme was monomeric. The cofactor Ca 2؉ was required for dimerization. Covalent modification of the active site serine with hexadecylsulfonylfluoride resulted in stabilization of the dimeric form and a loss of the absolute calcium requirement for dimerization. The results of these experiments provide evidence for dimerization as the molecular mechanism by which the enzymatic activity of OMPLA is regulated. This dimerization probably plays a role in vivo as well. Data from chemical cross-linking on whole cells indicate that OMPLA is present in the outer membrane as a monomer and that activation of the enzyme induces dimerization concurrent with the appearance of enzymatic activity.Outer membrane phospholipase A (OMPLA 1 ; also known as detergent-resistant phospholipase A or PldA protein) is one of the few enzymes present in the outer membrane of Gramnegative bacteria. OMPLA hydrolyzes the acyl ester bonds in (phospho)lipids and has Ca 2ϩ as an essential cofactor (1, 2). Initially, the Escherichia coli enzyme was purified and characterized (3), and its structural gene, designated pldA, was subsequently cloned and overexpressed (4 -7). Recently, Brok et al. (8) reported the cloning of the pldA genes of several other Enterobacteriaceae species. Comparison of the OMPLA amino acid sequences revealed a high degree of homology within this family, but no clear homology exists with sequences of watersoluble (phospho)lipases. A ␤-barrel structure has been proposed for OMPLA (8), analogous to the outer membrane porins, of which the x-ray structures have been solved (9 -11). Recently, we succeeded in the overproduction, in vitro refolding, and subsequent purification of the enzyme on a large scale (12), which allowed its crystallization (13).Although OMPLA is embedded in its own substrate in the cell envelope, no enzymatic activity can be detected in normally growing cells (14,15). Because OMPLA is expressed constitutively, genetic regulation cannot explain the absence of enzymatic activity. Moreover, the expressed protein is correctly transported to and inserted into the outer membrane, where it resides in a dormant state. Activity is induced by various processes that perturb the membrane, such as phage-induced lysis (16), temperature shock (4), and colicin secretion (17, 18). Similar results have been reported in vitro after reconstitution of OMPLA in lipid vesicles (19). Memb...

Substrate interferes with dimerisation of outer membrane phospholipase A

“…Previous work demonstrated that excess of the detergent C 12 SB leads to dissociation of dimeric OMPLA and subsequent loss in apparent initial enzymatic activity [9]. Thus, after preincubation at 20 mM C 12 SB only 10% residual initial activity was retained.…”

“…In£uence of assay parameters on lag phases and enzymatic activity Previously, it was shown that the experimental parameters strongly determine OMPLA activity [9]. Our current activity model Eq.…”

Bacterial phospholipase A: structure and function of an integral membrane phospholipase

“…In vitro, the phospholipase activity is modulated in a calcium dependent manner by reversible dimerisation. Inhibition by hexadecanesulphonyl £uoride stabilises the dimeric form of OMPLA [38]. In vivo, chemical cross-linking studies indicate the presence of the monomeric form of OMPLA in the outer membrane [38].…”

“…Inhibition by hexadecanesulphonyl £uoride stabilises the dimeric form of OMPLA [38]. In vivo, chemical cross-linking studies indicate the presence of the monomeric form of OMPLA in the outer membrane [38]. Soni¢cation or induction of bacteriocin release protein activates OMPLA which is a process concurring with dimerisation [39].…”

“…They are in fact competitive inhibitors with dissociation constants in the millimolar range [8]. In vitro, calcium is required for dimerisation [38]. Kinetic experiments indicate the in£u-ence of the detergent on the calcium a¤nity.…”