Canvasing the Substrate-Binding Pockets of the Wax Ester Synthase.

Abstract

The biosynthesis of wax esters and triglycerides in bacteria is accomplished through the action of the wax ester synthase/acyl-coenzyme A:diacylglycerol acyltransferase (WS/DGAT or wax ester synthase). A hallmark of these enzymes is the broad substrate profile that accepts alcohols, diglycerides, and fatty acyl-CoAs of various carbon chain lengths and degrees of branching. These enzymes have a broad biotechnological potential due to their role in producing high-value lipids or simple fuels similar to biodiesel through biosynthetic routes. Recently, a crystal structure was solved for the wax ester synthase from Marinobacter aquaeolei VT8 (Maqu_0168), providing a much clearer picture of the architecture of this enzyme and enabling a more precise analysis of the important structural features of the protein. In this work, we used the structure to canvas amino acids lining the proposed substrate-binding pockets and tested the effects of exchanging specific residues on the substrate profiles. We also developed an approach to better probe the residues that alter fatty acyl-CoA selectivity, which has proven more difficult to investigate. Our findings provide an improved blueprint for future efforts to understand how these enzymes position substrates for catalysis and to tailor or improve these enzymes in future biosynthetic schemes.