Biochemical and structural analysis of peroxisomal enzymes involved in ether phospholipid synthesis

Ether phospholipids are essential components of eukaryotic cell membrane and are involved in many fundamental functions such as bone development, signal transduction and intracellular transport. They are characterised by an alkyl or alkenyl substituent at sn-1 position of the glycerol backbone. Dihydroxyacetone-phosphate acyltransferase (DHAPAT) and the flavoenzyme alkyldihydroxyacetone-phosphate synthase (ADPS) work in complex to introduce an ether linkage into a phospholipid-precursor molecule. Deficit in ether phospholipids has very serious pathological consequences as observed in patients affected by genetic diseases such as Zellweger syndrome and rhizomelic chondrodysplasia punctata (RCDP). Here, we present the crystal structure of the mammalian Cavia porcellus ADPS, which reveals a dimeric organization. ADPS consists of a FAD-binding domain, a cap domain, and a substrate-binding domain. This third domain exhibits a beta-sheet and a gating alpha-helix, which is involved in the closure of a substrate tunnel specific for aliphatic chains of 16 carbon atoms. Superposition of C. porcellus and Dictyostelium discoideum ADPS structures shows that the FAD-binding domain is highly conserved between the two species whereas the substrate-binding domain is rotated by 20 degrees. Mutations in the amino acids Arg419, Tyr515, and Tyr578 have been found in patients affected by RCDP. Mutagenesis studies indicate that each of these mutations lead to the complete inactivation of the enzyme. Structural data confirmed the role of these side chains as they are located in the active site and are likely to directly take part in catalysis. Our data are discussed in the context of a proposed catalytic mechanism that involves formation of a covalent adduct between the flavin and the substrate, which enables the enzyme to catalyze the acyl-alkyl exchange reaction through a most unusual non-redox mechanism.