Discovery of a molecular caliper mechanism for determining the length of very long-chain fatty acids

Despite their numerous essential functions, our understanding of how very long-chain fatty acids (VLCFAs) are made was highly limited. From an academic standpoint, the chain length diversity of VLCFAs, which enables their functional diversity, represents a fascinating example of how polymerization of simple building blocks can be used to build chemically complex macromolecules. From a practical standpoint, there are potential therapeutic and commercial benefits to be gained from manipulating VLCFA synthesis. A key obstacle to these endeavors has been the fact that VLCFAs are made by a collection of detergent-labile, multi-enzyme complexes embedded in the ER membrane. Consequently, even though the basic outline of the chemistry underlying VLCFA synthesis was established in the 1960’s, the minimal set of proteins, including the key dehydratase component, needed for VLCFA synthesis was not known.

We identified Yjl097w (Phs1p) as the founding member of a new family of membrane proteins that act as the VLCFA dehydratase. This missing piece allowed us to reconstitute VLCFA synthesis in proteoliposomes containing purified Phs1p and three other membrane proteins. We used this in vitro system to address what is arguably the most important question regarding VLCFA synthesis: how does the biosynthetic machinery instruct the precise number of two-carbon additions that yields a defined length product. Our studies revealed a fascinating mechanism, akin to a caliper, in which the chain length is determined by the distance between the cytosolic active site that mediates two-carbon addition and a lysine near the luminal end of a transmembrane helix. By stepping this lysine residue along one face of the helix toward the cytosol, we engineered novel synthases with correspondingly shorter VLCFA outputs.