Investigating an unusual bacterial acyl carrier protein: a direct link between fatty acid and sphingolipid biosynthesis?

Sphingolipids are essential structural components of membranes found in all eukaryotes and have also been identified in some bacteria. The first step of sphingolipid biosynthesis pathway is catalysed by serine palmitoyltransferase (SPT), a member of the alpha‐oxoamine synthase (AOS) family, which catalyses a Claisen‐like condensation of L‐serine and palmitoyl‐coenzyme A to produce 3‐ketodihydrosphingosine (KDS). All SPT's and other members of the AOS family studied to date utilise an acyl‐CoA as substrate during this reaction. However, a sphingolipid‐producing bacterium, Sphingomonas wittichii , may utilise a small type II acyl carrier protein (ACP) to deliver the acyl chain to its SPT target. Sequencing of the S. wittichii genome allowed identification of a small gene encoding a putative ACP immediately upstream of the SPT gene. Converting the apo‐ACP to the substrate form, acyl‐ACP, has proved difficult. Amino acid sequence alignment, combined with modelling studies revealed an unusual tryptophan residue that could prevent modification to the acyl‐ACP form. Here we use a combination of techniques such as fluorescence and absorption spectroscopy, site‐directed mutagenesis and mass spectrometry to further characterise the S. wittichii ACP. We use chemical cross‐linking along with mass spectrometry to investigate protein‐protein interactions between the SPT and the ACP substrate. This research is funded by The Derek Stewart Charitable Trust, The University of Edinburgh, the BBSRC and the EPSRC.