IDENTIFICATION OF A NOVEL FAMILY OF BACTERIAL LIPOLYTIC ENZYMES

Lipolytic enzymes belong to a large class of enzymes called serine hydrolases that are distinguished by a presence of a nucleophilic serine (nSer) in their active site. Most of the bacterial lipolytic enzymes have the nSer located in a conservative GXSXG pentapeptide (X – any amino acid residue), GDSL motive or less frequently in a motive specific to lipolytic enzymes structurally resembling $\beta$-lactamases / DD-peptidases [1]. Conservative amino acid motives and biochemical properties of bacterial lipolytic enzymes are used for their classification [2]. In this work, a putative carboxylesterase (EstAG1) from Staphylococcus saprophyticus AG1 [3] with previously uncharacterized pentapeptide and nSer location motive was identified. Enzyme having no close homologues had unconventional GDGTG pentapeptide and nSer located in a SPXYD (Y – hydrophobic residue) sequence previously unspecified for the bacterial lipolytic enzymes. The putatively catalytic nSer and other amino acids related to catalytic function were investigated and validated by site-directed mutagenesis and activity analysis of the mutant recombinant enzymes. Phylogenetic analysis revealed that EstAG1 homologues are found in the other Staphylococcus spp. as well. Based on the low amino acid sequence identities, unique conservative amino acid motives and phylogenetic analysis results, EstAG1 belongs to a new family of bacterial lipolytic enzymes.