Deconstructing the DGAT1 enzyme: membrane interactions at substrate binding sites.

Jose L S Lopes,Ana P U Araujo,Leila M Beltramini,Bonnie A Wallace,Robert J Deschenes

doi:10.1371/journal.pone.0118407

Jose L S Lopes, Ana P U Araujo + Show 3 more

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https://doi.org/10.1371/journal.pone.0118407

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Journal: PloS one	Publication Date: Feb 26, 2015
Citations: 35	License type: CC BY 4.0

Affiliation: University of London

Abstract

Diacylglycerol acyltransferase 1 (DGAT1) is a key enzyme in the triacylglyceride synthesis pathway. Bovine DGAT1 is an endoplasmic reticulum membrane-bound protein associated with the regulation of fat content in milk and meat. The aim of this study was to evaluate the interaction of DGAT1 peptides corresponding to putative substrate binding sites with different types of model membranes. Whilst these peptides are predicted to be located in an extramembranous loop of the membrane-bound protein, their hydrophobic substrates are membrane-bound molecules. In this study, peptides corresponding to the binding sites of the two substrates involved in the reaction were examined in the presence of model membranes in order to probe potential interactions between them that might influence the subsequent binding of the substrates. Whilst the conformation of one of the peptides changed upon binding several types of micelles regardless of their surface charge, suggesting binding to hydrophobic domains, the other peptide bound strongly to negatively-charged model membranes. This binding was accompanied by a change in conformation, and produced leakage of the liposome-entrapped dye calcein. The different hydrophobic and electrostatic interactions observed suggest the peptides may be involved in the interactions of the enzyme with membrane surfaces, facilitating access of the catalytic histidine to the triacylglycerol substrates.

Highlights

Diacylglycerol acyltransferase (DGAT) is an enzyme that catalyses the esterification of a 1,2-diacylglycerol with a fatty acyl-CoA [1], resulting in a triacylglycerol (TAG) molecule in a reaction that is known to be the main limiting step in the TAG synthesis pathway [2].Homologous Diacylglycerol acyltransferase 1 (DGAT1) genes have been identified in a wide range of eukaryotic organisms, including yeast, plants, fungi, invertebrates and mammals
The fluorescence emission spectra of these peptides in aqueous solution confirmed the exposure of the Trp residues to the aqueous environment, since maximum emission was centered at 354 nm, a wavelength corresponding to the Trp emission when free in aqueous solution [27]
The use of the model membrane systems allowed us to distinguish between different modes of interaction of the predicted peptides involved in DGAT1 activity

Summary

Introduction

Diacylglycerol acyltransferase (DGAT) is an enzyme that catalyses the esterification of a 1,2-diacylglycerol with a fatty acyl-CoA [1], resulting in a triacylglycerol (TAG) molecule in a reaction that is known to be the main limiting step in the TAG synthesis pathway [2]. Homologous DGAT1 genes have been identified in a wide range of eukaryotic organisms, including yeast, plants, fungi, invertebrates and mammals. DGAT1 gene silencing reduced by ~50% the oil content of mature seeds [3]. In Drosophila, the DGAT1 gene was identified as causing premature apoptosis [4]. In addition to the clinical interest in the DGAT1 enzyme as a target for obesity treatment [5,6,7], studies have found related roles for this.

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