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Biochem Biophys Res Commun. 2019 Apr 30;512(2):310-313. doi: 10.1016/j.bbrc.2019.03.057. Epub 2019 Mar 16.

An alternative splicing alters the product outcome of a class I terpene synthase in Isodon rubescens.

Author information

1
College of Pharmacy, Hubei University of Chinese Medicine, Wuhan, 430065, China; State Key Laboratory Breeding Base of Dao-di Herbs, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, 100700, China. Electronic address: jblhandan@163.com.
2
State Key Laboratory Breeding Base of Dao-di Herbs, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, 100700, China. Electronic address: guojuanzy@163.com.
3
State Key Laboratory Breeding Base of Dao-di Herbs, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, 100700, China. Electronic address: jinfutang@126.com.
4
State Key Laboratory Breeding Base of Dao-di Herbs, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, 100700, China. Electronic address: tongyuru13@126.com.
5
State Key Laboratory Breeding Base of Dao-di Herbs, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, 100700, China. Electronic address: xiaoma1110@126.com.
6
State Key Laboratory Breeding Base of Dao-di Herbs, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, 100700, China. Electronic address: chentong_biology@163.com.
7
State Key Laboratory Breeding Base of Dao-di Herbs, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, 100700, China. Electronic address: wyn19870127@126.com.
8
State Key Laboratory Breeding Base of Dao-di Herbs, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, 100700, China. Electronic address: niffay0502@163.com.
9
State Key Laboratory Breeding Base of Dao-di Herbs, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, 100700, China. Electronic address: diana1989119@sina.com.
10
State Key Laboratory Breeding Base of Dao-di Herbs, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, 100700, China. Electronic address: laichangjiang44@126.com.
11
State Key Laboratory Breeding Base of Dao-di Herbs, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, 100700, China. Electronic address: guanghongcui@163.com.
12
College of Pharmacy, Hubei University of Chinese Medicine, Wuhan, 430065, China; State Key Laboratory Breeding Base of Dao-di Herbs, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, 100700, China. Electronic address: huangluqi01@126.com.

Abstract

The labdane-related diterpenoids are an important superfamily of natural products. Their structural diversity mainly depends on diterpene synthases, which generate the hydrocarbon skeletal structures. Isodon rubescens contains an expanded family of class I terpene synthases with different functions. Here we report a novel class I terpene synthase cDNA (IrKSL3a) with loss of 18 nucleotides compared with the reported cDNA sequence (IrKSL3). Inspection of IrKSL3 genomic sequence indicated that IrKSL3a and IrKSL3 transcripts may be generated by an alternative splicing event that utilizes different 3' splice site. In vitro assays showed that IrKSL3a produced isopimaradiene and miltiradiene, while IrKSL3 only produced miltiradiene. Protein sequence alignment found the six residues encoded by the alternative exon was unique to IrKSL3, which are 17 residues away from the conserved DDXXD motif. A deletion mutant of IrKSL3 showed that maintaining two residues within the six-amino acid is sufficient for miltiradiene production, while the other mutants lost nearly all enzymatic function. Our results illustrated how product outcomes can be changed by alternative splicing, and further gave an interesting example for studying the loop conformation in tuning product outcome in class I terpene synthase.

KEYWORDS:

Alternative splicing; Class I terpene synthase; Isodon rubescens

PMID:
30890335
DOI:
10.1016/j.bbrc.2019.03.057

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