Unusual Catalytic Triad of Escherichia coli Outer Membrane Phospholipase A

Abstract

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.

Crystal structure and catalytic mechanism of the LPS 3-O-deacylase PagL fromPseudomonas aeruginosa

“…Third, mutagenesis showed that Glu-140 is important for PagL activity, although not essential, because residual activity was detected after its substitution by Ala. In OMPLA, Asn-156 is not essential either, because Asn156Ala substitution reduced OMPLA activity Ϸ20-fold (18). A possible explanation is that OMPLA, and probably also PagL, has a highly functional oxyanion hole that can partly compensate for the loss of the acidic residue.…”

TheBacillus subtilisSignaling Protein SpoIVB Defines a New Family of Serine Peptidases

“…In some systems, however, the Asp simply serves to maintain the correct position of the active site histidine. Indeed, in cysteine proteases, a conserved Asn residue (e.g., Asn175 in papain) serves this very purpose (35) and Asn156 appears in the hydrolase catalytic triad of E. coli outer membrane phospholipase A (15). We assume that the role of Asp363 is to stabilize the appropriate His263 rotamer or tautomer in SpoIVB catalysis, a function that could potentially be performed by Asn.…”

Substrate interferes with dimerisation of outer membrane phospholipase A

“…Initial OMPLA activities at various calcium concentrations could be ¢tted to a hyperbolic saturation curve to yield a calcium a⁄nity of 18 WM [17]. The ¢nal OMPLA activities obtained with Eq.…”