As hydrophobic residues make up

As hydrophobic residues make up almost 40% of all the residues in the DGAT1 proteins [74] (Fig. 2a and c), they presumably play an important role in facilitating the reaction. While it is clear that alterations within the Sit1 motif, especially of the critical tyrosine (Y), alter binding to oleoyl-CoA and reduce enzyme activity, further evidence is needed to ascertain whether this effect is due to the disruption of the phosphorylation site or a result of alteration of the hydrophobicity in this region brought about by replacing the tyrosine and tryptophan residues with amphiphilic amino acids. The arginine (R) residue of the catalytic motif RLIIEN in plant DGAT1 is thought to accept a proton from the hydroxyl group of DAG to allow a nucleophilic attack of the thioester bond of acyl-CoA [91]. The importance of the hydrophobicity in the regions flanking the putative catalytic sites has been demonstrated by Xu et al. [92] who were able to abolish DGAT1 activity in Tropaeolum majus by mutagenesis of the hydrophobic phenylalanine (F) to an arginine (R) in the putative DAG binding site. A well-characterized two nucleotide substitution, located in (S)-10-Hydroxycamptothecin mg 8 of bovine DGAT1, changes a lysine (K) to an alanine (K232A). The amount of TAG synthesized with the K allele is almost 1.5 times the amount synthesized with the A allele [93]. This mutation affects the TAG content of milk in spite of being upstream of the Sit1 and Sit2 motifs found from 356FGDREFYRDWWNSES370 and 379NIPVHKWSIRHFY391, respectively. The two binding sites, possibly aided by other luminal motifs, may act in concert using a combination of hydrophobic and electrostatic interactions to bring the substrates into close proximity near the membrane where histidine in the proposed active site can catalyze the formation of TAG [94]. It has also been shown that a synthetic peptide with the same sequence as the Sit2 motif can interact with negatively-charged surfaces, and this may facilitate the binding of DGAT1 to its substrates and/or the ER membrane [88]. The interaction of the Sit2 domain with a charged domain in a biological membrane could bring about configuration changes that facilitate molecular recognition by enabling the enzyme to interact with the membrane surface where the substrates for TAG synthesis, DAGs and fatty acyl CoAs, are localized [87,94]. The importance of conformational changes in enzyme activity was demonstrated even before the DGAT activity was attributed to discrete DGAT isoforms. It has been reported that the N-terminus of DGAT1 is responsible for DGAT1 tetramer formation and that the C-terminus is dispensable [95]. Prevention of tetramer formation is accompanied by a several-fold increase in in vitro enzyme activity, indicating that the dimer may be the quaternary structure suitable for catalysis and that switching between the two forms may have a regulatory significance [77]. Although these efforts partially shed light on the mechanisms of action of DGAT1 enzyme, similar to DGAT2, the three-dimensional structure is needed to fully understand its enzymatic function.