Outer membrane phospholipase A (OMPLA) is an integral membrane enzyme that catalyses the hydrolysis of phospholipids. Enzymatic activity is regulated by reversible dimerisation and calcium-binding. We have investigated the role of calcium by X-ray crystallography. In monomeric OMPLA, one calcium ion binds between two external loops (L3L4 site) at 10 A Ê from the active site. After dimerisation, a new calcium-binding site (catalytic site) is formed at the dimer interface in the active site of each molecule at 6 A Ê from the L3L4 calcium site. The close spacing and the difference in calcium af®nity of both sites suggests that the L3L4 site may function as a storage site for a calcium ion, which relocates to the catalytic site upon dimerisation. A sequence alignment demonstrates conservation of the catalytic calcium site but evolutionary variation of the L3L4 site. The residues in the dimer interface are conserved as well, suggesting that all outer membrane phospholipases require dimerisation and calcium in the catalytic site for activity. For this family of phospholipases, we have characterised a consensus sequence motif (YTQ-X n -G-X 2 -H-X-SNG) that contains conserved residues involved in dimerisation and catalysis.
Escherichia coli outer membrane phospholipase A (OMPLA) is an integral membrane enzyme. OMPLA is active as a homodimer and requires calcium as a cofactor. The crystal structures of the monomeric and the inhibited dimeric enzymes were recently determined [Snijder, H. J., et al. (1999) Nature 401, 717-721] and revealed that OMPLA monomers are folded into a 12-stranded antiparallel beta-barrel. The active site consists of previously identified essential residues Ser144 and His142 in an arrangement resembling the corresponding residues of a serine hydrolase catalytic triad. However, instead of an Asp or Glu that normally is present in the triad of serine hydrolases, a neutral asparagine (Asn156) was found in OMPLA. In this paper, the importance of the catalytic Asn156 is addressed by site-directed mutagenesis studies. All variants were purified at a 30 mg scale, and were shown to be properly folded using SDS-PAGE and circular dichroism spectroscopy. Using chemical cross-linking, it was shown that all variants were not affected in their calcium-dependent dimerization properties. The Asn156Asp variant exhibited a 2-fold lower activity than wild-type OMPLA at neutral pH. Interestingly, the activity of the variant is 1 order of magnitude higher than that of the wild type at pH >10. Modest residual activities (5 and 2.5%, respectively) were obtained for the Asn156Ala and Asn156Gln mutants, showing that the active site of OMPLA is more tolerant toward replacements of this third residue of the catalytic triad than other serine hydrolases, and that the serine and histidine residues are minimally required for catalysis. In the X-ray structure of dimeric OMPLA, the cofactor calcium is coordinating the putative oxyanion via two water molecules. We propose that this may lessen the importance for the asparagine in the catalytic triad of OMPLA.
The activity of outer membrane phospholipase A (OMPLA) is regulated by reversible dimerization. However, native OMPLA reconstituted in phospholipid vesicles was found to be present as a dimer but nevertheless inactive. To investigate the importance of dimerization for control of OMPLA activity, a covalent OMPLA dimer was constructed and its properties were compared to native OMPLA both in a micellar detergent and after reconstitution in a phospholipid bilayer. Unlike native OMPLA, activity of the covalent OMPLA dimer was independent of type and concentration of detergent in micellar systems. In such systems, the covalent OMPLA dimer invariantly displayed high calcium affinity. In contrast, high calcium concentrations were required to activate a covalent OMPLA dimer when present in intact vesicles. Solubilization of the vesicles increased the affinity for calcium, suggesting that in an intact bilayer the dimer interface is not properly formed. This was supported by the observation that OMPLA variants having an impaired dimeric interface also lacked high affinity calcium binding. A covalent linkage was not able to restore high affinity calcium binding in these variants, demonstrating that a proper dimer interface is essential for optimal catalysis.Keywords: OMPLA; dimerization; calcium binding; activity regulation.The outer membrane phospholipase A (OMPLA) is an integral membrane enzyme that catalyses the hydrolysis of acylester bonds in phospholipids using calcium as a cofactor [1]. The enzyme is widespread among Gram-negative bacteria, both in pathogens and nonpathogens. In pathogenic bacteria such as Campylobacter coli and Helicobacter pylori OMPLA is involved in pathogenesis and virulence [2,3]. In nonpathogenic bacteria the physiological function of OMPLA is less clear. The Escherichia coli enzyme has been best studied and is involved in the secretion of bacteriocins, antibacterial peptides that are produced in order to survive under starvation conditions [4,5].Although OMPLA is constitutively expressed, no phospholipid turnover can be detected under physiological conditions, suggesting that OMPLA resides in the outer membrane in an inactive state. OMPLA activity can be triggered by processes that severely perturb the outer membrane integrity, such as phage-induced lysis [6], spheroplast formation [7], heat shock [8], EDTA treatment [9] and colicin release [4,10,11].In vitro OMPLA activity is strongly dependent on the experimental conditions such as type of detergent, detergent concentration and protein concentration. It has been shown that activity is regulated by reversible dimerization and that the experimental conditions influence the dimerization equilibrium [12]. Chemical cross-linking on whole cells indicated that OMPLA is present in the outer membrane as a monomer, and that activation by bacteriocin-release protein-induced dimerization [5]. This suggests that in vivo dimerization is also part of the regulatory mechanism. However, fluorescence resonance energy transfer experiments on OMPLA reconstitute...
Outer membrane phospholipase A (OMPLA) from Escherichia coli is an integral-membrane enzyme with a unique His-Ser-Asn catalytic triad. In serine proteases and serine esterases usually an Asp occurs in the catalytic triad; its role has been the subject of much debate. Here the role of the uncharged asparagine in the active site of OMPLA is investigated by structural characterization of the Asn156Ala mutant. Asparagine 156 is not involved in maintaining the overall active-site configuration and does not contribute significantly to the thermal stability of OMPLA. The active-site histidine retains an active conformation in the mutant notwithstanding the loss of the hydrogen bond to the asparagine side chain. Instead, stabilization of the correct tautomeric form of the histidine can account for the observed decrease in activity of the Asn156Ala mutant.
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