Anandamide amidohydrolase of porcine brain: cDNA cloning, functional expression and site-directed mutagenesis

Anandamide (arachidonoylethanolamide) is an endogenous ligand for cannabino id receptors, and its cannabimimetic activities are lost when the compound is hydrolyzed to arachidonic acid and ethanolamine by an enzyme referred to as anandamide amidohydrolase. We cloned a cDNA for the enzyme of porcine b rain, and the cDNA encoded a protein of 579 amino acids with a molecular ma ss of 62.9 kDa. The amino acid sequence was 81, 80 and 85% identical with t he enzymes previously cloned from the liver of rat, mouse, and human, respe ctively. When the enzyme protein was overexpressed in COS-7 cells, the part iculate fraction of the cells showed an anandamide hydrolyzing activity and also catalyzed the reverse reaction synthesizing anandamide from arachidon ic acid and ethanolamine both with a specific activity of 0.2-0.3 mu mol/mi n/mg protein at 37 degrees C. The brain enzyme exhibited a wide substrate s pecificity hydrolyzing oleamide, 2-arachidonoylglycerol, and methyl arachid onate. The point mutation of Ser-217, Asp-237, Ser-241, or Cys-249 complete ly abolished the hydrolyses of all the above-mentioned substrates as well a s the synthesis of anandamide in the reverse reaction. (C) 1999 Elsevier Sc ience B.V. All rights reserved.