Both histidine residues of the conserved HHXXXDG motif are essential for wax ester synthase/acyl-CoA:diacylglycerol acyltransferase catalysis

Bacterial acyltransferases of the wax ester synthase/diacylglycerol acyltransferase (WS/DGAT) family possess a highly conserved HHXXXDG motif. In this study, we describe the first experimental evidence that this motif is part of the active site of WS/DGAT from the Acinetobacter baylyi strain ADP1 and that it is crucial for enzymatic activity. The second histidine residue of this motif (H₁₃₃) turned out to be essential for the catalytic activity. In addition, the replacement of the first histidine (His₁₃₂) also led to explicitly decreased activity. A complete loss of activity was only observed upon substitution of both histidine residues by leucine, revealing that both are necessary for maximal activity. In contrast, the replacement of Asp₁₃₇ and Gly₁₃₈ against alanine had only little effect on enzyme activity, thus demonstrating that they are not essential for WS/DGAT catalysis although belonging to the highly conserved motif. One peculiarity of WS/DGAT enzymes is their little substrate specificity regarding hydrophobic compounds. In this study, we demonstrated the inability of WS/DGAT to accept polar compounds as substrates.