Functional characterization of a peroxisomal acyltransferase gene family

Sammanfattning: Peroxisomes are cell organelles that play an important role in the metabolism of a variety of lipids including very long chain fatty acids, dicarboxylic acids, bile acids, and xenobiotic carboxylic acids. The metabolism of lipids in peroxisomes requires the concerted efforts of many different enzymes, which includes members of the type I acyl-CoA thioesterase/acyltransferase protein family and the nudix hydrolases. We have identified two new peroxisomal members of the thioesterase/acyltransferase protein family, which we have named acyl-CoA:amino acid N-acyltransferase 1 (ACNAT1) and 2 (ACNAT2). ACNAT1 acts as an acyltransferase that efficiently conjugates long-chain and very long-chain fatty acids to taurine; these novel metabolites are known as N-acyltaurines (NATs) and can allow the excretion of fatty acids as taurine conjugates, or alternatively these NAT may have roles in cell signaling. While we have not yet established an activity for ACNAT2 we hypothesize that it also functions as an acyltransferase, but with different substrate specificity to ACNAT1. The type I acyl-CoA thioesterase/acyltransferase family contains nine members in total that display two different types of activities, one is hydrolysis of fatty acyl-CoAs and the other is the conjugation of bile acids or fatty acids to glycine and/or taurine. These proteins belong to the !/" hydrolase family in which a highly conserved structure, called the nucleophilic elbow is very important for activity. By using site directed mutagenesis we demonstrate the importance of the amino acids in the nucleophilic elbow for activity, and propose a hypothesis for the evolution of this gene family. Coenzyme A (CoASH) is an important cofactor in peroxisomal metabolic pathways. Peroxisomes contain their own pool of CoASH and an unresolved issue is how this pool is regulated. It has been suggested that nudix hydrolase 7! (NUDT7!) regulates the peroxisomal CoASH pool by metabolizing CoASH. However, our study shows that NUDT7! is mainly active towards a wide range of acyl-CoAs, with highest activity towards medium-chain acyl-CoAs and lower activity with CoASH. This suggests that NUDT7!, together with the acyl-CoA thioesterases, has a function in the regulation of peroxisomal acyl-CoA/CoASH levels.