The type-I acyl-CoA thioesterase/acyltransferase gene family : linking structure to function

Abstract

Acyl-CoA thioesterases hydrolyse acyl-CoAs to non-esterified fatty acids and coenzyme A (CoASH), thereby regulating intracellular levels of these compounds. We have recently identified and initiated characterisation of a novel, unique gene family of acyl-CoA thioesterases/acyltransferases, called the Type-I acyl-CoA thioesterase/acyltransferase gene family. In mouse this gene family comprises of six acyl-CoA thioesterase genes (Acot1-6) organised in one cluster, and the bile acid-CoA: amino acid N-acyltransferase (BACAT) gene and two putative peroxisomal thioesterase genes (PTE-le and -11) organised in a second cluster.

All these genes show a high degree of conservation, and the aim of this thesis work was to characterise the structure/function relationship that defines esterase or acyltransferase activity among these family members. Initial secondary structure prediction and site-directed mutagenesis experiments identified Acot1 as an esterase belonging to the alpha/beta (CL/P) hydrolase family of enzymes, and based on the homology within this gene family it follows that all members belong to this family of proteins. The alpha/beta hydrolases are characterised by a highly conserved catalytic machinery consisting of a nucleophilic residue (usually a Ser), a general base (His) and an acidic residue (Asp or Glu). The nucleophilic residue is located in a highly conserved structure known as the nucleophilic elbow within a consensus motif of GlyXaaSerXaaGly. Interestingly, the members of the Type-I gene family display three variants of this nucleophilic elbow motif, Acot1-6 that contain the common elbow motif (GlyXaaSerXaaGly), PTE-le and -If that contain the motif SerXaaSerXaaGly and BACAT that contains the motif SerXaaCysXaaGly.

In this thesis work, I have characterised one enzyme with each elbow motif (Acot1, BACAT and PTE-Ie) and show that the variations in the nucleophilic elbow are crucial for the esterase and acyltransferase activities of these enzymes. The data show that Acot1-6 are indeed acyl-CoA thioesterases, while BACAT and PTE-le act as acyltransferases, thus identifying different functions for the two gene clusters. Interestingly, the nucleophilic elbow of BACAT contains two nucleophiles (the Ser and Cys residues) that mediate thioesterase and acyltransferase activity, respectively. Based on these results and sequence analysis, the diverged structures and functions of the enzymes of this gene family are discussed from an evolutionary perspective.