Crystal Structure of the Bifunctional Wax Synthase 1 from Acinetobacter baylyi Suggests a Conformational Change upon Substrate Binding and Formation of Additional Substrate Binding Sites

Abstract

Wax esters (WE) are neutral lipids that are formed by the transesterification of an activated fatty acyl moiety to a fatty alcohol. Due to their diverse physicochemical properties, WE are used as industrial lubricants, in cosmetics, or for coating. There is substantial interest in producing WE in bacteria and plants by genetic engineering to improve their sustainability and to reduce production costs. However, we lack a detailed understanding of the catalytic mechanism and structural determinants that influence substrate specificities of WE-synthesizing enzymes, which is essential for tailored WE production. One class of well-studied WE-producing enzymes are the bifunctional bacterial wax synthases/acyl-CoA:diacylglycerol O-acyltransferases (WSD). Here, we report the 1.95 Å crystal structure of Acinetobacter baylyi WSD1 (AbWSD1) with a fatty acid molecule bound in the active site. The location of a cocrystallized myristic acid confirms a previously proposed acyl-CoA binding site. A comparison of this AbWSD1 structure to a published Marinobacter aquaeolei WSD1 (MaWSD1) structure of the apoenzyme revealed a major structural difference in the C-terminal part of AbWSD1. This leads us to propose a conformational change in AbWSD1 induced by substrate binding. This conformational change forms then a potential coenzyme A (CoA) binding site. Furthermore, we have identified an additional cavity in AbWSD1 and could show through mutational studies that two amino acids lining the cavity are crucial for the acyl-CoA:diacylglycerol O-acyltransferase (DGAT) activity of the enzyme. Our findings provide a foundation for designing WSD variants that lack DGAT activity.