Regulating the activity and selectivity of squalene-hopene-cyclases via the membrane anchor

• Applicant: Professor Dr. Bernhard Hauer
• Subject Area: Biochemistry
• Term: since 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 524081117

Consideration of the complex membrane environment is great importance to the study of membrane-bound enzymes but leads to difficulties when studying the enzymes in isolation. Although new methods have increased the accessibility of membrane-bound enzymes as part of a complex multicomponent system, there is still an asymmetry in our knowledge to the detriment of these proteins. An example of this knowledge gap is the lack in our understanding of how membrane-bound enzymes are regulated by different membrane properties such as composition, geometry, or biochemical and physical factors. To elucidate a part of this interaction this project will investigate the membrane anchor of monotopic proteins as a possible regulatory element. As a model system to study this element of the enzymatic structure, we consider the monotopic triterpene cyclase squalene-hopene cyclase (SHC). These enzymes control membrane fluidity by producing highly hydrophobic hopanoids to counteract biological stress on the host organism. Therefore, a mutual influence of membrane fluidity and enzyme activity of SHCs seems comprehensible. Supporting this hypothesis we observed in previous studies, that the promiscuous cyclization of (E,E)-homofarnesol to (-)-ambrox in the in vivo system was subject to substrate-excess inhibition, which was not observed for the purified enzyme. Thus, it can be speculated that local membrane perturbations caused by terpenes, result in an inhibitory effect on the activity of SHCs. To investigate the role of the anchor motif in this interaction, we plan to generate a focused collection of rational SHC variants whose modifications focus on this motif and the surrounding amino acids. These variants will first be selected for indications of alterations in membrane interaction using a series of model reactions and subsequently characterized by introduction into E. coli or mycoplasmas with modified membrane composition. The aim is to investigate whether the modification of the membrane anchor affects or even completely uncouples the interaction between the membrane and enzymatic activity, thus identifying the membrane anchor as a mediator between membrane and catalytic apparatus. Indeed, prior reconstruction of the Free Energy Landscape of the wildtype and a laboratory-evolved variant showed that different conformations of this α-helix could be displayed by the different SHC variants when they are embedded on the lipid membrane. To support these studies, in addition to characterization in the laboratory, further computational studies of the generated variants will also be performed taking into account varying membrane system to rationalize the influence of the introduced mutations. A deeper understanding of these relationships would add another element to our understanding of membrane proteins and could allow us to draw conclusions on the regulation of other monotopic membrane proteins.

• DFG Programme: Research Grants