Two Predicted Transmembrane Domains Exclude Very Long Chain Fatty acyl-CoAs from the Active Site of Mouse Wax Synthase.

Steffen Kawelke,Ivo Feussner

doi:10.1371/journal.pone.0145797

Abstract

Wax esters are used as coatings or storage lipids in all kingdoms of life. They are synthesized from a fatty alcohol and an acyl-CoA by wax synthases. In order to get insights into the structure-function relationships of a wax synthase from Mus musculus, a domain swap experiment between the mouse acyl-CoA:wax alcohol acyltransferase (AWAT2) and the homologous mouse acyl-CoA:diacylglycerol O-acyltransferase 2 (DGAT2) was performed. This showed that the substrate specificity of AWAT2 is partially determined by two predicted transmembrane domains near the amino terminus of AWAT2. Upon exchange of the two domains for the respective part of DGAT2, the resulting chimeric enzyme was capable of incorporating up to 20% of very long acyl chains in the wax esters upon expression in S. cerevisiae strain H1246. The amount of very long acyl chains in wax esters synthesized by wild type AWAT2 was negligible. The effect was narrowed down to a single amino acid position within one of the predicted membrane domains, the AWAT2 N36R variant. Taken together, we provide first evidence that two predicted transmembrane domains in AWAT2 are involved in determining its acyl chain length specificity.

Highlights

Wax esters (WEs) are used as coatings or beside triacylglycerols (TAGs) as storage lipids in all kingdoms of life [1]
In order to analyze if the similarity of diacylglycerol O-acyltransferase 2 (DGAT2) and AWAT2 on their sequence level is reflected by their domain structure, three different programs (Phobius [18], TMHMM [16] and SOSUI [17]) were used for the prediction of the domain structure of AWAT2
As DGAT2, AWAT2 is predicted to contain an N-terminal cytoplasmic tail that is linked to two TM domains, which are connected by a short linker (Fig 2A)

Summary

Introduction

Wax esters (WEs) are used as coatings or beside triacylglycerols (TAGs) as storage lipids in all kingdoms of life [1]. Two enzymes are needed for WE synthesis: a fatty acyl reductase (FAR) and a wax synthase (WS). The first enzyme, FAR, provides the acyl acceptor moiety of a WE through formation of a fatty alcohol via a NAD(P)H-dependent reduction of a fatty acyl-CoA molecule [4]. This reaction releases a first CoA molecule.

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Conclusion