Abstract
The molybdoflavoenzymes (MFEs), aldehyde oxidase (AOX) and xanthine oxidoreductase (XOR) catalyse the oxidation of many drugs, environmental pollutants and N-heterocyclic compounds. MFEs are enzymes that contain flavin adenine dinucleotide (FAD), iron sulphur domain [2Fe-2S] and molybdenum cofactor domain (Moco). This research study examined the structural evolution of MFEs in chordates and the effects of polymorphisms on vertebrate MFEs. Numerous databases such as NCBI were used multiple alongside alignment software such as CLC Sequence Viewer and UGENE.The species-specific motifs identified in MFEs included an extra twenty three amino acids found in Fe/S domain in birds and reptiles, nine extra amino acids in mammalian AOX2 and three amino acids specific to mammalian XDH only. Analysis of the evolutionary change in the primary amino acid sequence of XDH has not been done previously in chordates. In addition, scrutiny of the amino acids in vertebrate AOX isoforms, reveals the relationship between primitive vertebrate AOXs such as AOXα, AOXβ and the ancestral vertebrate precursor XDH. Scrutiny of the amino acids near the active site of XDH in non-mmmalian vertebrate species demonstrated that amino acids around the NAD binding site, particularly Tyr 393 (human XDH) and the purine binding site such as Glu803 and Arg881 (human XDH) are conserved in all chordates. In addition, significant amino acids changes in XDH in vertebrates during evolution were studied. Two cysteines (Cys535 and Cys992) residues that are crucial in the conversion of XDH to XO in rats and cow were analysed in all chordates. These were conserved in all mammals except monotremes but were not conserved in other chordate classes. Two other cysteines involved in XDH to XO conversion (Cys1318 and Cys1326) located at C-terminus were also examined. Cys1318 was found in all vertebrates except Sea lamprey but Cys1326 was found in mammals only.
Human XDH polymorphisms such as (Asn11Lys, Asn909Lys, Thr910Met, Cys1318Tyr, Glu133Lys and Glu133Lys) and AOX polymorphisms such as (Arg921His, Asn1135Ser and His1297Arg) were scrutinized and found to be conserved in all chordates including C. elegans (nematode worm) and in Drosophila melanogaster (fruit fly). These polymorphisms were also studied regarding the impacts on structure of MFEs.
Previous studies in this laboratory showed no phenanthridine oxidation activity in Sprague Dawley rat and Fischer rat strains using a spectrophotometric assay. Using a sensitive HPLC assays it was found that Wistar rat had the highest activity towards AOX substrates such as aldehydes and N-heterocycilc compounds, in contrast to the other strains. Structural examination of the polymorphism acids (Gly110Ser and Ala852Val) in AOX1 and Gln39, Val 852, Gln937, and Met1078 in rat strains revealed a basis for the loss of activity in Sprgue Dawley and Fischer rat.
To evaluate if Drosophila melanogaster and C. elegans could be used as experimental models for MFEs, they were tested for their ability to catalyse the oxidation of a number of AOX and XDH substrates. Vanillin and xanthine was used to evaluate MFE activity in Drosophila and for the first time in C. elegans. The results demonstrated that Drosophila and C. elegans were able to catalyse the oxidation of vanillin. Spectrophotometric and HPLC assays demonstrated that dimethylaminocinnamaldehyde (DMAC), vanillin, phenanthridine, and xanthine were substrates for MFEs in these organisms.
Structural features of D. melanogaster and C. elegans MFEs were compared with their wellresearched mammalian counterparts. Arg334, Trp335, Arg426, and Phe549 in bovine XDH that are conserved in all vertebrates are not conserved in Drosophila melanogaster and Caenorhabditis elegans. However, investigation of Glu802, Arg880 and Glu1261 (bovine XDH numbering) revealed that these residues are conserved in Drosophila melanogaster and Caenorhabditis elegans as well as all chordates.
| Date of Award | 2 Sep 2019 |
|---|---|
| Original language | English |
| Supervisor | Dougie Clarke (Main Supervisor) & Roger Phillips (Co-Supervisor) |