Literature summary for 4.2.3.44 extracted from

Yang, R.; Du, Z.; Qiu, T.; Sun, J.; Shen, Y.; Huang, L.
Discovery and functional characterization of a diverse diterpene synthase family in the medicinal herb Isodon lophanthoides var. gerardiana (2021), Plant Cell Physiol., 62, 1423-1435.

Cloned(Commentary)

Cloned (Comment)	Organism
DNA and amino acid sequence determination and analysis, phylogenetic analysis, recombinant expression of wild-type and mutant enzymes in Saccharomyces cerevisiae strain YS100, subcloning in Escherichia coli strain TOP10, transient heterologous expression of wild-type and mutant enzymes in Nicotiana benthaminana via transfection with Agrobacterium tumefaciens strain LBA4404, quantitative RT-PCR enzyme expression analysis	Isodon lophanthoides var. gerardianus

Cloned (Comment)

Organism

DNA and amino acid sequence determination and analysis, phylogenetic analysis, recombinant expression of wild-type and mutant enzymes in Saccharomyces cerevisiae strain YS100, subcloning in Escherichia coli strain TOP10, transient heterologous expression of wild-type and mutant enzymes in Nicotiana benthaminana via transfection with Agrobacterium tumefaciens strain LBA4404, quantitative RT-PCR enzyme expression analysis

Isodon lophanthoides var. gerardianus

Protein Variants

Protein Variants	Comment	Organism
A513I	site-directed mutagenesis, substitution of A513 with Ile completely alters the product from nezukol to isopimara-7,15-diene	Isodon lophanthoides var. gerardianus
A513S	site-directed mutagenesis, mutant IlKSL5A513S mutant predominantly produces nezukol, like the wild-type enzyme	Isodon lophanthoides var. gerardianus
A513T	site-directed mutagenesis, mutant IlKSL5A513T produces a mixture of nezukol and isopimara-7,15-diene	Isodon lophanthoides var. gerardianus
A513V	site-directed mutagenesis, mutant IlKSL5A513V produces predominantly isopimar-7,15-diene with trace amount of nezukol	Isodon lophanthoides var. gerardianus
additional information	the hydrophobicity and the size of amino acid 513 play an important role in determining the addition of water by IlKSL5. The Ile and Val mutants with relatively large aliphatic side chains may change the substrate conformation and shield the carbocation of the pimar-15-en-8-yl+ intermediate, thus preventing the addition of a water molecule. By contrast, Ala and other small and/or hydrophilic residues, e.g. Ser and Thr may allow the contact between the pimar-15-en-8-yl+ intermediate and a water molecule, leading to the carbocation neutralization through the addition of a water molecule to form nezukol. In addition, mutation of IrTPS Ala 523 that corresponds to IlKSL5 A513 to Ile shows the same effect by altering product form nezukol to isopimar-7,15-diene. Product comparison with the parallel product of a monofunctional isopimara-7,15-diene synthase from Pinus contorta (PcmISO1)	Isodon lophanthoides var. gerardianus

Organism

Organism	UniProt	Comment	Textmining
Isodon lophanthoides var. gerardianus	A0A8F6T6B3	plants are collected from the Yaowang Mountain in Guangzhou University of Chinese Medicine (113°E, 23°N) on April 2019	-

Source Tissue

Source Tissue	Comment	Organism	Textmining
leaf	-	Isodon lophanthoides var. gerardianus	-
root	-	Isodon lophanthoides var. gerardianus	-
stem	-	Isodon lophanthoides var. gerardianus	-

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
(+)-copalyl diphosphate	-	Isodon lophanthoides var. gerardianus	isopimara-7,15-diene + diphosphate	-	?
additional information	diterpene profiling of plant tissues (root periderms, root cortexes and xylems, stems and leaves) and compound identification by GC-MS analysis	Isodon lophanthoides var. gerardianus	?	-	-

Synonyms

Synonyms	Comment	Organism
IlKSL5	-	Isodon lophanthoides var. gerardianus
kaurene synthase-like 5	-	Isodon lophanthoides var. gerardianus
additional information	see also EC 4.2.3.183	Isodon lophanthoides var. gerardianus

General Information

General Information	Comment	Organism
evolution	the enzyme belongs to the class I diterpene synthases (DiTPSs). Phylogenetic tree, joining trees of Isodon lophanthoides diTPSs with known class II and class I enzymes based on aligned protein sequences, overview. IlKSL5 belongs to a sub-clade involved in specialized metabolism	Isodon lophanthoides var. gerardianus
malfunction	the Ile and Val mutants with relatively large aliphatic side chains may change the substrate conformation and shield the carbocation of the pimar-15-en-8-yl+ intermediate, thus preventing the addition of a water molecule. By contrast, Ala and other small and/or hydrophilic residues, e.g. Ser and Thr may allow the contact between the pimar-15-en-8-yl+ intermediate and a water molecule, leading to the carbocation neutralization through the addition of a water molecule to form nezukol. In addition, mutation of IrTPS Ala 523 that corresponds to IlKSL5 A513 to Ile shows the same effect by altering product from nezukol to isopimar-7,15-diene	Isodon lophanthoides var. gerardianus
metabolism	biosynthetic pathway of specialized and general metabolism of diterpenoids in medicinal plant Isodon lophanthoides var. gerardiana, overview. Diverse diTPSs producing distinct diterpene scaffolds form the foundation of the diterpene chemical diversity. Diterpene scaffolds are further modified by P450s and other modification enzymes	Isodon lophanthoides var. gerardianus
additional information	the hydrophobicity and the size of amino acid 513 play an important role in determining the addition of water by IlKSL5. To identify the amino acid residue controlling the water addition of enzyme IlKSL5, homology-based structure models of IlKSL5 are created using the crystal structure of abietadiene synthase from Abies grandis (PDB ID 3S9V) as template	Isodon lophanthoides var. gerardianus