Alkyl-dihydroxyacetonephosphate Synthase. Presence and Role of Flavin Adenine Dinucleotide

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.

Persistence of duplicated PAC1 receptors in the teleost, Sparus auratus

Background: Duplicated genes are common in vertebrate genomes. Their persistence is assumed to be either a consequence of gain of novel function (neofunctionalisation) or partitioning of the function of the ancestral molecule (sub-functionalisation). Surprisingly few studies have evaluated the extent of such modifications despite the numerous duplicated receptor and ligand genes identified in vertebrate genomes to date. In order to study the importance of function in the maintenance of duplicated genes, sea bream (Sparus auratus) PAC(1) receptors, sequence homologues of the mammalian receptor specific for PACAP (Pituitary Adenylate Cyclase-Activating Polypeptide), were studied. These receptors belong to family 2 GPCRs and most of their members are duplicated in teleosts although the reason why both persist in the genome is unknown. Results: Duplicate sea bream PACAP receptor genes (sbPAC(1)A and sbPAC(1)B), members of family 2 GPCRs, were isolated and share 77% amino acid sequence identity. RT-PCR with specific primers for each gene revealed that they have a differential tissue distribution which overlaps with the distribution of the single mammalian receptor. Furthermore, in common with mammals, the teleost genes undergo alternative splicing and a PAC(1)Ahop1 isoform has been characterised. Duplicated orthologous receptors have also been identified in other teleost genomes and their distribution profile suggests that function may be species specific. Functional analysis of the paralogue sbPAC(1)s in Cos7 cells revealed that they are strongly stimulated in the presence of mammalian PACAP(27) and PACAP(38) and far less with VIP (Vasoactive Intestinal Peptide). The sbPAC(1) receptors are equally stimulated (LOG(EC50) values for maximal cAMP production) in the presence of PACAP27 (-8.74 +/- 0.29 M and -9.15 +/- 0.21 M, respectively for sbPAC(1)A and sbPAC(1)B, P > 0.05) and PACAP(38) (-8.54 +/- 0.18 M and -8.92 +/- 0.24 M, respectively for sbPAC(1)A and sbPAC(1)B, P > 0.05). Human VIP was found to stimulate sbPAC(1)A (-7.23 +/- 0.20 M) more strongly than sbPAC(1)B (-6.57 +/- 0.14 M, P < 0.05) and human secretin (SCT), which has not so far been identified in fish genomes, caused negligible stimulation of both receptors. Conclusion: The existence of functionally divergent duplicate sbPAC(1) receptors is in line with previously proposed theories about the origin and maintenance of duplicated genes. Sea bream PAC(1) duplicate receptors resemble the typical mammalian PAC(1), and PACAP peptides were found to be more effective than VIP in stimulating cAMP production, although sbPAC(1)A was more responsive for VIP than sbPAC(1)B. These results together with the highly divergent pattern of tissue distribution suggest that a process involving neofunctionalisation occurred after receptor duplication within the fish lineage and probably accounts for their persistence in the genome. The characterisation of further duplicated receptors and their ligands should provide insights into the evolution and function of novel protein-protein interactions associated with the vertebrate radiation