An esterase from Escherichia coli with a sequence similarity to hormone-sensitive lipase

An esterase from Escherichia coli that is a member of the hormone-sensitive lipase (HSL) family was overproduced, purified and characterized. It is encoded by the ybaC gene and composed of 319 amino acid residues with an M-r of 36038. The enzymic activity was determined by using various p-nitrophenyl esters of fatty acids as a substrate at 25 degrees C and pH 7.1. The enzyme showed hydrolytic activity towards substrates with an acyl chain length of less than 8, whereas it showed little hydrolytic activity towards those with an acyl chain length of more than 10. In addition, it showed little hydrolytic activity towards trioleoylglycerol and cholesterol oleate. Determination of the kinetic parameters for the hydrolyses of the substrates from C-2 to C-8 indicates that C-4 and C-5 substrates are the most preferred. Close agreement between the M,determined by SDS/PAGE (37000) and column chromatography (38000) suggests that the enzyme exists in a monomeric form. It is an acidic protein with a pi value of 4.1. The far-UV CD spectrum suggests that its helical content is 26.1 %. Comparison of the amino acid sequence of this enzyme with those involved in the HSL family allows us to propose that Ser(165), Asp(262) and His(292) constitute the catalytic triad of E. coli esterase.