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

Abstract

ORCID IDs: 0000-0001-8699-690X (R.J.W.); 0000-0003-4745-9153 (M.J.L.).Diacylglycerol acyltransferase 1 (DGAT1) is an integral membrane enzyme catalyzing the final and committed step in the acylcoenzyme A (CoA)-dependent biosynthesis of triacylglycerol (TAG). The biochemical regulation of TAG assembly remains one of the least understood areas of primary metabolism to date. Here, we report that the hydrophilic N-terminal domain of Brassica napus DGAT1 (BnaDGAT1 1-113 ) regulates activity based on acyl-CoA/CoA levels. The N-terminal domain is not necessary for acyltransferase activity and is composed of an intrinsically disordered region and a folded segment. We show that the disordered region has an autoinhibitory function and a dimerization interface, which appears to mediate positive cooperativity, whereas the folded segment of the cytosolic region was found to have an allosteric site for acyl-CoA/CoA. Under increasing acyl-CoA levels, the binding of acyl-CoA with this noncatalytic site facilitates homotropic allosteric activation. Enzyme activation, on the other hand, is prevented under limiting acyl-CoA conditions (low acyl-CoA-to-CoA ratio), whereby CoA acts as a noncompetitive feedback inhibitor through interaction with the same folded segment. The three-dimensional NMR solution structure of the allosteric site revealed an a-helix with a loop connecting a coil fragment. The conserved amino acid residues in the loop interacting with CoA were identified, revealing details of this important regulatory element for allosteric regulation. Based on these results, a model is proposed illustrating the role of the N-terminal domain of BnaDGAT1 as a positive and negative modulator of TAG biosynthesis.

Properties and Biotechnological Applications of Acyl‐CoA:diacylglycerol Acyltransferase and Phospholipid:diacylglycerol Acyltransferase from Terrestrial Plants and Microalgae

“…In addition, the N‐terminal region of B. napus DGAT1 formed dimers and tetramers based on crosslinking experiments (Weselake et al, ). Consistently, analysis of mouse and B. napus DGAT1 showed that the N‐terminal region plays a role in self‐oligomerization (Caldo et al, ; McFie et al, ). Furthermore, the hydrophilic N‐terminal region of B. napus DGAT1 was shown to constitute the enzyme's regulatory domain, which is not necessary for catalysis (Caldo et al, ).…”

“…Consistently, analysis of mouse and B. napus DGAT1 showed that the N‐terminal region plays a role in self‐oligomerization (Caldo et al, ; McFie et al, ). Furthermore, the hydrophilic N‐terminal region of B. napus DGAT1 was shown to constitute the enzyme's regulatory domain, which is not necessary for catalysis (Caldo et al, ). This domain is comprised of two distinct segments, specifically an intrinsically disordered region (IDR) and a folded segment (Fig.…”

“…The recent insights into DGAT structure–function relationships largely rely on the identification of possible functional motifs and the determination of putative membrane topologies. Very recently, the structure of the hydrophilic N‐terminal domain of DGAT1 from B. napus (BnaDGAT1) was solved, which resulted in a leap forward in gaining insight into the biochemical regulation of this enzyme family (Caldo et al, ). Below, the structural and functional features of plant DGAT with relevant reference to the mammalian literature are discussed.…”

“…This observation agrees with a previous study on mouse DGAT1, wherein removal of N‐terminal fragments led to increased normalized enzyme activity (McFie et al, ). The solution NMR structure of the folded segment of the N‐terminal region of B. napus DGAT1 showed that it is composed of an α ‐helix near the first predicted TMD (Caldo et al, ). Loops and coils connected this helix to the IDR.…”

“…Loops and coils connected this helix to the IDR. The loop near the α ‐helix was shown to contain the allosteric site for acyl‐CoA and CoA, which serves as a homotropic activator and a feedback inhibitor of the enzyme, respectively (Caldo et al, ). The small‐angle X‐ray scattering structure of this domain showed that the monomer has a highly extended structure, exhibiting various heterogeneous conformations.…”