Deconstructing the DGAT1 enzyme: Binding sites and substrate interactions

Abstract

Diacylglycerol acyltransferase 1 (DGAT1) is a microsomal membrane enzyme responsible for the final step in the synthesis of triacylglycerides. Although DGATs from a wide range of organisms have nearly identical sequences, there is little structural information available for these enzymes. The substrate binding sites of DGAT1 are predicted to be in its large luminal extramembranous loop and to include common motifs with acyl-CoA cholesterol acyltransferase enzymes and the diacylglycerol binding domain found in protein kinases. In this study, synthetic peptides corresponding to the predicted binding sites of DGAT1 enzyme were examined using synchrotron radiation circular dichroism spectroscopy, fluorescence emission and adsorption onto lipid monolayers to determine their interactions with substrates associated with triacylglyceride synthesis (oleoyl-CoA and dioleoylglycerol). One of the peptides, Sit1, which includes the FYxDWWN motif common to both DGAT1 and acyl-CoA cholesterol acyltransferase, changes its conformation in the presence of both substrates, suggesting its capability to bind their acyl chains. The other peptide (Sit2), which includes the putative diacylglycerol binding domain HKWCIRHFYKP found in protein kinase C and diacylglycerol kinases, appears to interact with the charged headgroup region of the substrates. Moreover, in an extended-peptide which contains Sit1 and Sit2 sequences separated by a flexible linker, larger conformational changes were induced by both substrates, suggesting that the two binding sites may bring the substrates into close proximity within the membrane, thus catalyzing the formation of the triacylglyceride product.

Diacylglycerol Acyltransferase 1 Is Regulated by Its N-Terminal Domain in Response to Allosteric Effectors

“…Numerals indicate the locations of the different enzyme truncation points. S1 and S2 correspond to the acylCoA and diacylglycerol binding regions in the active site, respectively, which were identified in bovine DGAT1 (Lopes et al, 2014). The putative catalytic His (H), identified in murine DGAT1, also is shown in the topology (McFie et al, 2010).…”

“…That study also demonstrated the importance of the N-terminal region in forming dimers and tetramers and hypothesized the role of this region in enzyme regulation. The active site region in bovine DGAT1 was characterized through secondary structure analysis and binding studies of short peptide fragments spanning the predicted substrate binding sites (Lopes et al, 2014). The N and C termini of tung tree (Vernicia fordii) DGAT1 were both localized in the cytosol, and a C-terminal endoplasmic reticulum (ER) retrieval motif was shown to target the plant enzyme to the ER (Shockey et al, 2006).…”

Deconstructing the DGAT1 Enzyme: Membrane Interactions at Substrate Binding Sites

“…Substrate binding sites in bovine DGAT1 enzyme that are identical to portions of the ACAT and protein kinase C forms enzymes have been identified [17]. The diacylglycerol (DAG) binding site is located in a soluble extramembranenous loop of the protein, rather than in a transmembrane domain, suggesting it would interact with DAG headgroup on (or close to) the membrane surface [17, 18].…”

“…The diacylglycerol (DAG) binding site is located in a soluble extramembranenous loop of the protein, rather than in a transmembrane domain, suggesting it would interact with DAG headgroup on (or close to) the membrane surface [17, 18]. Synthetic peptides (Sit1 and Sit2) corresponding to these proposed substrate binding sites of bovine DGAT1 [17] were shown to interact with the substrates for triacylglycerol synthesis (DAGs and acyl-CoAs) which are present in the membrane. Consequently, it is proposed that the peptides would need to interact with the membrane, in order to access and present the substrates to the catalytic histidine [19] which is located in the same extramembranous loop.…”

Interactions of bioactive molecules & nanomaterials with Langmuir monolayers as cell membrane models

“…For DOG and OCoA in equal proportions at the interface, SIT2 cannot penetrate into the hydrophobic region, remaining in contact with the lipid polar head. For small proportions of OCoA in DOG or DOG in OCoA, Lopes et al[38] designed three peptides to investigate DGAT binding sites and substrate interactions: the first one, named Sit1, includes the most conserved region in DGAT1, the FYxDWWN segment. Sit2 is the putative diacylcgycerol binding domain, and includes the sequence HKWCIRHFYKP.…”