Alkyl-dihydroxyacetonephosphate synthase - Presence and role of flavin adenine dinucleotide

Alkyl-dihydroxyacetonephosphate synthase is a peroxisomal enzyme involved i n ether lipid synthesis, It catalyzes the exchange of the acyl chain in acy l-dihydroxy acetonephosphate for a long chain fatty alcohol, yielding the f irst ether linked intermediate, i.e. alkyl-dihydroxyacetonephosphate, in th e pathway of ether lipid biosynthesis. Although this reaction is not a net redox reaction, the amino acid sequence of the enzyme suggested the presenc e of a flavin adenine dinucleotide (FAD)-binding domain. In this study we s how that alkyl-dihydroxy acetonephosphate synthase contains an essential FA D molecule as cofactor, which is evidenced by fluorescence properties, UV-v isible absorption spectra and the observation that the enzyme activity is d ependent on the presence of this cofactor in a coupled in vitro transcripti on/translation assay. Furthermore, we could demonstrate that the FAD cofact or directly participates in catalysis. Upon incubation of the enzyme with t he substrate palmitoyl-dihydroxy acetonephosphate, the flavin moiety is red uced, indicating that in this initial step the substrate is oxidized. Stopp ed flow experiments show that the reduction of the flavin moiety is a monop hasic process yielding a oxygen stable, reduced enzyme species. Upon additi on of hexadecanol to the reduced enzyme species, the flavin moiety is effic iently reoxidized. A hypothetical reaction mechanism is proposed that is co nsistent with the data in this paper and with previous studies.