Amplification and bioinformatics analysis of conserved FAD-binding region of L-amino acid oxidase (LAAO) genes in gastropods compared to other organisms

This study aimed to investigate the conserved FAD-binding region of the L-amino acid oxidase (LAAO) genes in twelve gastropod genera commonly found in Thailand compared to those in other organisms using molecular cloning, nucleotide sequencing and bioinformatics analysis. Genomic DNA of gastropods and other invertebrates was extracted and screened using primers specific to the conserved FAD-binding region of LAAO. The amplified 143-bp fragments were cloned and sequenced. The obtained nucleotide sequences of 21 samples were aligned and phylogenetically compared to the LAAO-conserved FAD-binding regions of 210 other organisms from the NCBI database. Translated amino acid sequences of these samples were used in phylogenetics and pattern analyses. The phylogenetic trees showed clear separation of the conserved regions in fungi, invertebrates, and vertebrates. Alignment of the conserved 47-amino-acid FAD-binding region of the LAAOs showed 150 unique sequences among the 231 samples and these patterns were different from those of other flavoproteins in the amine oxidase family. An amino acid pattern analysis of five sub-regions (bFAD, FAD, FAD-GG, GG, and aGG) within the FAD-binding sequence showed high variation at the FAD-GG sub-region. Pattern analysis of secondary structures indicated the aGG sub-region as having the highest structural variation. Cluster analysis of these patterns revealed two major clusters representing the mollusc clade and the vertebrate clade. Thus, molecular phylogenetics and pattern analyses of sequence and structural variations could reflect evolutionary relatedness and possible structural conservation to maintain specific function within the FAD-binding region of the LAAOs in gastropods compared to other organisms. Keywords: L-amino acid oxidase (LAAO), FAD-binding region, Gastropods, Pattern analysis, Clustering