Yasotha Sundaraj and Hasdianty Abdullah and Nima Ghahremani Nezhad and Kenneth Francis Rodrigues and Suriana Sabri and Syarul Nataqain Baharum (2023) Cloning, expression and functional characterization of a novel a-humulene synthase, responsible for the formation of sesquiterpene in agarwood originating from Aquilaria malaccensis. Current Issues in Molecular Biology, 45 (11). pp. 1-15.
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Abstract
This study describes the cloning, expression and functional characterization of α-humulene synthase, responsible for the formation of the key aromatic compound α-humulene in agarwood originating from Aquilaria malaccensis. The partial sesquiterpene synthase gene from the transcriptome data of A. malaccensis was utilized for full-length gene isolation via a 30 RACE PCR. The complete gene, denoted as AmDG2, has an open reading frame (ORF) of 1671 bp and encodes for a polypeptide of 556 amino acids. In silico analysis of the protein highlighted several conserved motifs typically found in terpene synthases such as Asp-rich substrate binding (DDxxD), metal-binding residues (NSE/DTE), and cytoplasmic ER retention (RxR) motifs at their respective sites. The AmDG2 was successfully expressed in the E. coli:pET-28a(+) expression vector whereby an expected band of about 64 kDa in size was detected in the SDS-PAGE gel. In vitro enzyme assay using substrate farnesyl pyrophosphate (FPP) revealed that AmDG2 gave rise to two sesquiterpenes: α-humulene (major) and β-caryophyllene (minor), affirming its identity as α-humulene synthase. On the other hand, protein modeling performed using AlphaFold2 suggested that AmDG2 consists entirely of α-helices with short connecting loops and turns. Meanwhile, molecular docking via AutoDock Vina (Version 1.5.7) predicted that Asp307 and Asp311 act as catalytic residues in the α-humulene synthase. To our knowledge, this is the first comprehensive report on the cloning, expression and functional characterization of α-humulene synthase from agarwood originating from A. malaccensis species. These findings reveal a deeper understanding of the structure and functional properties of the α-humulene synthase and could be utilized for metabolic engineering work in the future.
| Item Type: | Article |
|---|---|
| Keyword: | α-humulene synthase; sesquiterpene; Aquilaria malaccensis; protein modeling; molecular docking |
| Subjects: | Q Science > QK Botany > QK1-989 Botany > QK710-899 Plant physiology S Agriculture > SD Forestry > SD1-669.5 Forestry > SD391-410.9 Sylviculture |
| Department: | INSTITUTE > Biotechnology Research Institute (BRI) |
| Depositing User: | ABDULLAH BIN SABUDIN - |
| Date Deposited: | 20 Feb 2024 11:40 |
| Last Modified: | 20 Feb 2024 11:40 |
| URI: | https://eprints.ums.edu.my/id/eprint/38318 |
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