STRUCTURE OF A MICROBIAL HOMOLOG OF MAMMALIAN PLATELET-ACTIVATING-FACTOR ACETYLHYDROLASES - STREPTOMYCES EXFOLIATUS LIPASE AT 1.9 ANGSTROM RESOLUTION

Background: Neutral lipases are ubiquitous and diverse enzymes. The mo lecular architecture of the structurally characterized lipases is simi lar, often despite a lack of detectable homology at the sequence level . Some of the microbial lipases are evolutionarily related to physiolo gically important mammalian enzymes. For example, limited sequence sim ilarities were recently noted for the Streptomyces exfoliatus lipase ( SeL) and two mammalian platelet-activating factor acetylhydrolases (PA F-AHs). The determination of the crystal structure of Set allowed us t o explore the structure-function relationships in this novel family of homologous hydrolases. Results: The crystal structure of Set was dete rmined by multiple isomorphous replacement and refined using data to 1 .9 Angstrom resolution. The molecule exhibits the canonical tertiary f old of an alpha/beta hydrolase, The putative nucleophilic residue, Ser 131, is located within a nucleophilic elbow and is hydrogen bonded to His209, which in turn interacts with Asp177. These three residues crea te a triad that closely resembles the catalytic triads found in the ac tive sites of other neutral lipases. The mainchain amides of Met132 an d Phe63 are perfectly positioned to create an oxyanion hole. Unexpecte dly, there are no secondary structure elements that could render the a ctive site inaccessible to solvent, like the lids that are commonly fo und in neutral lipases, Conclusions: The crystal structure of Set rein forces the notion that it is a homologue of the mammalian PAF-AHs, We have used the catalytic triad in Set to model the active site of the P AF-AHs, Our model is consistent with the site-directed mutagenesis stu dies of plasma PAF-AH, which implicate Ser273, His351 and Asp296 in th e active site. Our study therefore provides direct support for the hyp othesis that the plasma and isoform II PAF-AHs are triad-containing al pha/beta hydrolases.