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Int J Mol Sci
2024 Nov 29;2523:. doi: 10.3390/ijms252312881.
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Structure of Genes Encoding Oxidosqualene Cyclases-Key Enzymes of Triterpenoid Biosynthesis from Sea Cucumber Eupentacta fraudatrix.
Baldaev SN
,
Chausova VE
,
Isaeva KV
,
Boyko AV
,
Stonik VA
,
Isaeva MP
.
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Oxidosqualene cyclases (OSCs) are enzymes responsible for converting linear triterpenes into tetracyclic ones, which are known as precursors of other important and bioactive metabolites. Two OSCs genes encoding parkeol synthase and lanostadienol synthase have been found in representatives of the genera Apostichopus and Stichopus (family Stichopodidae, order Synallactida). As a limited number of sea cucumber OSCs have been studied thus far, OSCs encoding gene(s) of the sea cucumber Eupentacta fraudatrix (family Sclerodactylidae, order Dendrochirotida) were investigated to fill this gap. Here, we employed RACEs, molecular cloning, and Oxford Nanopore Technologies to identify candidate OSC mRNAs and genes. The assembled cDNAs were 2409 bp (OSC1) and 3263 bp (OSC2), which shared the same CDS size of 2163 bp encoding a 721-amino-acid protein. The E. fraudatrix OSC1 and OSC2 had higher sequence identity similarity to each other (77.5%) than to other holothurian OSCs (64.7-71.0%). According to the sequence and molecular docking analyses, OSC1 with L436 is predicted to be parkeol synthase, while OSC2 with Q439 is predicted to be lanostadienol synthase. Based on the phylogenetic analysis, E. fraudatrix OSCs cDNAs clustered with other holothurian OSCs, forming the isolated branch. As a result of gene analysis, the high polymorphism and larger size of the OSC1 gene suggest that this gene may be an ancestor of the OSC2 gene. These results imply that the E. fraudatrix genome contains two OSC genes whose evolutionary pathways are different from those of the OSC genes in Stichopodidae.
Figure 1. Alignment of OSCs proteins from sea cucumbers, sea urchin, starfish, and human. E. fraudatrix OSC1 and OSC2 (OR725688 and OR711403 in this study); A. japonicus LAS1 (PS) and LAS2 (LDS) (ON478352.1 and ON478353.1, respectively); A. parvimensis PS and LDS (ON478351.1 and ON478350.1); S. horrens OSC1 and OSC2 [14]; LSS: A. planci (XM 022227483.1), S. purpuratus (ON478349.1), H. sapiens (NM 002340.6). Conservative residues are in red colors. Active site residues are indicated by black circles. QXXXW motifs are marked on a consensus sequence by black boxes. LWIHCR and DTTAE motifs are marked by red boxes. Key residues, which determine enzyme function, are labeled with the blue box.
Figure 2. Molecular docking of triterpenoid in the active site of oxidosqualene cyclase. Functionally significant residues L436/L435 of EfOSC1 (a) and AjLAS1 (b) near the B and C rings of parkeol are shown in red. Functionally significant residues Q439/Q444 of EfOSC2 (c) and AjLAS2 (d) near the B ring of lanostadienol are shown by blue.
Figure 3. Maximum likelihood (ML) phylogenetic trees based on amino acid sequences of OSCs with (a) and without (b) sea cucumbers: H. sapiens (NM 002340.6), B. taurus (NM 001046564.1), M. musculus (XM 036155651.1), B. belcheri (XM 019790446.1), S. kowalevskii (XM 006825036.1), L. anatina (XM 013562424.1), L. gigantea (XM 009046756.1), P. canaliculata (XM 025240299.1), L. variegatus (XM 041622296.1), S. purpuratus (ON478349.1), P. miniata (ON478348.1), A. planci (XM 022227483.1), T. adhaerens (XM 002110738.1), A. queenslandica (XM 003383129.3), E. fraudatrix OSC1 (OR725688, this study), E. fraudatrix OSC2 (OR711403, this study), A. japonicus LAS1 (ON478352.1), A. japonicus LAS2 (ON478353.1), A. parvimensis PS (ON478351.1), A. parvimensis LDS (ON478350.1), S. horrens OSC1 [14], S. horrens OSC2 [14], D. discoideum (XM 641154.1), and S. cerevisiae (NP 011939.2).
Figure 4. Comparison of E. fraudatrix OSCs gene structures and their haplotypes. (a) Gene structure schemes. (b) Scatter plots of OSC1 and OSC2 transcripts (1), deduced amino acid sequences (2), and gene sequences (3) comparison. (c) Scatter plots of pairwise alignment through BLAST of four OSC1 gene haplotypes (comparison 1—haplotypes 1 and 2; 2—haplotypes 1 and 3; 3—haplotypes 1 and 4; 4—haplotypes 2 and 3; 5—haplotypes 2 and 4; 6—haplotypes 3 and 4). (d) Scatter plots of pairwise alignment through BLAST of four OSC2 gene haplotypes (comparison 1—haplotypes 1 and 2; 2—haplotypes 1 and 3; 3—haplotypes 1 and 4; 4—haplotypes 2 and 3; 5—haplotypes 2 and 4; 6—haplotypes 3 and 4).
