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

Abstract

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 (H 133 ) turned out to be essential for the catalytic activity. In addition, the replacement of the first histidine (His 132 ) 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 Asp 137 and Gly 138 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.

Use of Limited Proteolysis and Mutagenesis To Identify Folding Domains and Sequence Motifs Critical for Wax Ester Synthase/Acyl Coenzyme A:Diacylglycerol Acyltransferase Activity

“…5) that could be responsible for the WS/DGAT activity. In the HHxxxDG acyltransferases, the second histidine (His141 in Ma2) has been proposed to be the only catalytic residue that acts as a general base to extract the proton from the hydroxyl group of the alcohol to catalyze the nucleophilic attack (30), although both histidines were shown to be essential for wax ester synthase/acylCoA:diacylglycerol acyltransferase catalysis in A. baylyi (26). In Ma2, alanine scanning not only shows that the H141A mutant is poorly active in vitro but that it is one of the most deleterious mutants of all the mutants tested, thus confirming the essential catalytic role of the second histidine in WS/DGAT.…”

“…The main conserved motif in WS/ DGAT enzymes is the HHxxxDG motif, also present in other acylCoA-dependent acyltransferases ( Fig. 2A) (26).…”

“…All the studied WS/DGATs contain the conserved catalytic motif HHxxxDG, which has been proven to be involved in the protein activity (26). A similar HHxxxDG motif is conserved in a large number of acyl-CoAdependent acyltransferases also involved in the transfer of fatty acyl groups by the nucleophilic attack of a hydroxyl group on the thioester bond of the fatty acyl-CoA (13,(27)(28)(29)(30).…”

Acyltransferases in Bacteria

“…Furthermore, AtfA does not accept polar substrates such as sugars, organic acids, amino acids, naphthol, amines, or carotenoids. Its catalytic center might be located in a hydrophobic pocket or channel, restricting the accessibility for hydrophilic molecules (59).…”

“…Although aspartate and glycine are conserved amino acids in this motif, they seem not to be of major importance for the enzyme activity, because a replacement of either by alanine resulted in no significant decrease of enzyme activity. Their possible structural function still has to be elucidated (59).…”