title: Alkyl-dihydroxyacetonephosphate synthase. Presence and role of flavin adenine dinucleotide.
abstract: Alkyl-dihydroxyacetonephosphate synthase is a peroxisomal enzyme involved in ether lipid synthesis. It catalyzes the exchange of the acyl chain in acyl-dihydroxyacetonephosphate for a long chain fatty alcohol, yielding the first ether linked intermediate, i.e. alkyl-dihydroxyacetonephosphate, in the pathway of ether lipid biosynthesis. Although this reaction is not a net redox reaction, the amino acid sequence of the enzyme suggested the presence of a flavin adenine dinucleotide (FAD)-binding domain. In this study we show that alkyl-dihydroxyacetonephosphate synthase contains an essential FAD molecule as cofactor, which is evidenced by fluorescence properties, UV-visible absorption spectra and the observation that the enzyme activity is dependent on the presence of this cofactor in a coupled in vitro transcription/translation assay. Furthermore, we could demonstrate that the FAD cofactor directly participates in catalysis. Upon incubation of the enzyme with the substrate palmitoyl-dihydroxyacetonephosphate, the flavin moiety is reduced, indicating that in this initial step the substrate is oxidized. Stopped flow experiments show that the reduction of the flavin moiety is a monophasic process yielding a oxygen stable, reduced enzyme species. Upon addition of hexadecanol to the reduced enzyme species, the flavin moiety is efficiently reoxidized. A hypothetical reaction mechanism is proposed that is consistent with the data in this paper and with previous studies.
authors.affiliation: Centre for Biomembranes and Lipid Enzymology, Institute for Biomembranes, Utrecht University, 3584 CH Utrecht, The Netherlands.
authors.forename: E C
authors.initials: EC
authors.lastname: de Vet
authors.forename: Y H
authors.initials: YH
authors.lastname: Hilkes
authors.forename: M W
authors.initials: MW
authors.lastname: Fraaije
authors.forename: H
authors.initials: H
authors.lastname: van den Bosch
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000737:chemistry* / Q000235:genetics; D000818:Animals; D004099:Dihydroxyacetone Phosphate / Q000031:analogs & derivatives / Q000378:metabolism; D005486:Flavin Mononucleotide / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry* / Q000378:metabolism; D006168:Guinea Pigs; D007700:Kinetics; D016296:Mutagenesis; D010084:Oxidation-Reduction; D014176:Protein Biosynthesis; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D014158:Transcription, Genetic
chemical_list: D011994:Recombinant Proteins; D005182:Flavin-Adenine Dinucleotide; C003037:palmitoyl dihydroxyacetone phosphate; D004099:Dihydroxyacetone Phosphate; D005486:Flavin Mononucleotide; D019883:Alkyl and Aryl Transferases; C019654:alkylglycerone-phosphate synthase