Staff View: Cloning, expression and functional characterization of a novel a-humulene synthase, responsible for the formation of sesquiterpene in agarwood originating from Aquilaria malaccensis

Cloning, expression and functional characterization of a novel a-humulene synthase, responsible for the formation of sesquiterpene in agarwood originating from Aquilaria malaccensis

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. mal...

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Main Authors:	Yasotha Sundaraj, Hasdianty Abdullah, Nima Ghahremani Nezhad, Kenneth Francis Rodrigues, Suriana Sabri, Syarul Nataqain Baharum
Format:	Article
Language:	English English
Published:	ResearchGate 2023
Subjects:	QK710-899 Plant physiology SD391-410.9 Sylviculture
Online Access:	https://eprints.ums.edu.my/id/eprint/38318/1/ABSTRACT.pdf https://eprints.ums.edu.my/id/eprint/38318/2/FULL%20TEXT.pdf https://eprints.ums.edu.my/id/eprint/38318/ http://dx.doi.org/10.3390/cimb45110564
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spelling	my.ums.eprints.383182024-02-20T03:40:25Z https://eprints.ums.edu.my/id/eprint/38318/ Cloning, expression and functional characterization of a novel a-humulene synthase, responsible for the formation of sesquiterpene in agarwood originating from Aquilaria malaccensis Yasotha Sundaraj Hasdianty Abdullah Nima Ghahremani Nezhad Kenneth Francis Rodrigues Suriana Sabri Syarul Nataqain Baharum QK710-899 Plant physiology SD391-410.9 Sylviculture 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. ResearchGate 2023 Article NonPeerReviewed text en https://eprints.ums.edu.my/id/eprint/38318/1/ABSTRACT.pdf text en https://eprints.ums.edu.my/id/eprint/38318/2/FULL%20TEXT.pdf 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. http://dx.doi.org/10.3390/cimb45110564
institution	Universiti Malaysia Sabah
building	UMS Library
collection	Institutional Repository
continent	Asia
country	Malaysia
content_provider	Universiti Malaysia Sabah
content_source	UMS Institutional Repository
url_provider	http://eprints.ums.edu.my/
language	English English
topic	QK710-899 Plant physiology SD391-410.9 Sylviculture
spellingShingle	QK710-899 Plant physiology SD391-410.9 Sylviculture Yasotha Sundaraj Hasdianty Abdullah Nima Ghahremani Nezhad Kenneth Francis Rodrigues Suriana Sabri Syarul Nataqain Baharum Cloning, expression and functional characterization of a novel a-humulene synthase, responsible for the formation of sesquiterpene in agarwood originating from Aquilaria malaccensis
description	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.
format	Article
author	Yasotha Sundaraj Hasdianty Abdullah Nima Ghahremani Nezhad Kenneth Francis Rodrigues Suriana Sabri Syarul Nataqain Baharum
author_facet	Yasotha Sundaraj Hasdianty Abdullah Nima Ghahremani Nezhad Kenneth Francis Rodrigues Suriana Sabri Syarul Nataqain Baharum
author_sort	Yasotha Sundaraj
title	Cloning, expression and functional characterization of a novel a-humulene synthase, responsible for the formation of sesquiterpene in agarwood originating from Aquilaria malaccensis
title_short	Cloning, expression and functional characterization of a novel a-humulene synthase, responsible for the formation of sesquiterpene in agarwood originating from Aquilaria malaccensis
title_full	Cloning, expression and functional characterization of a novel a-humulene synthase, responsible for the formation of sesquiterpene in agarwood originating from Aquilaria malaccensis
title_fullStr	Cloning, expression and functional characterization of a novel a-humulene synthase, responsible for the formation of sesquiterpene in agarwood originating from Aquilaria malaccensis
title_full_unstemmed	Cloning, expression and functional characterization of a novel a-humulene synthase, responsible for the formation of sesquiterpene in agarwood originating from Aquilaria malaccensis
title_sort	cloning, expression and functional characterization of a novel a-humulene synthase, responsible for the formation of sesquiterpene in agarwood originating from aquilaria malaccensis
publisher	ResearchGate
publishDate	2023
url	https://eprints.ums.edu.my/id/eprint/38318/1/ABSTRACT.pdf https://eprints.ums.edu.my/id/eprint/38318/2/FULL%20TEXT.pdf https://eprints.ums.edu.my/id/eprint/38318/ http://dx.doi.org/10.3390/cimb45110564
_version_	1792152929649557504
score	13.211869

Cloning, expression and functional characterization of a novel a-humulene synthase, responsible for the formation of sesquiterpene in agarwood originating from Aquilaria malaccensis

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