Unusual catalytic triad of Escherichia coli outer membrane phospholipase A

Escherichia coli outer membrane phospholipase A (OMPLA) is an integral memb rane enzyme. OMPLA is active as a homodimer and requires calcium as a cofac tor. The crystal structures of the monomeric and the inhibited dimeric enzy mes were recently determined [Snijder, H. J., et al. (1999) Nature 401, 717 -721] and revealed that OMPLA monomers are folded into a 12-stranded antipa rallel beta-barrel. The active site consists of previously identified essen tial residues Ser144 and His142 in an arrangement resembling the correspond ing residues of serine hydrolase catalytic triad. However, instead of an As p or Glu that normally is present in the triad of serine hydrolases, a neut ral asparagine (Asn156) was found in OMPLA. In this paper, the importance o f the catalytic Asn156 is addressed by site-directed mutagenesis studies. A ll variants were purified at a 30 mg scale, and were shown to be properly f olded using SDS-PAGE and circular dichroism spectroscopy. Using chemical cr oss-linking, it was shown that all variants were not affected in their calc ium-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 wil d type at pH >10. Modest residual activities (5 and 2.5%, respectively) wer e obtained for the Asn156Ala and Asn156Gln mutants, showing that the active site of OMPLA is more tolerant toward replacements of this third residue o f the catalytic triad than other serine hydrolases, and that the serine and histidine residues are minimally required for catalysis. In the X-ray stru cture of dimeric OMPLA, the cofactor calcium is coordinating the putative o xyanion via two water molecules. We propose that this may lessen the import ance for the asparagine in the catalytic triad of OMPLA.