4.2.3.44	A513I	site-directed mutagenesis, substitution of A513 with Ile completely alters the product from nezukol to isopimara-7,15-diene	775070	
4.2.3.44	A513S	site-directed mutagenesis, mutant IlKSL5A513S mutant predominantly produces nezukol, like the wild-type enzyme	775070	
4.2.3.44	A513T	site-directed mutagenesis, mutant IlKSL5A513T produces a mixture of nezukol and isopimara-7,15-diene	775070	
4.2.3.44	A513V	site-directed mutagenesis, mutant IlKSL5A513V produces predominantly isopimar-7,15-diene with trace amount of nezukol	775070	
4.2.3.44	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)	775070	
4.2.3.44	additional information	transient heterologous expression of the isopimaric acid pathway in Nicotiana benthamiana. Expressing DXS and GGPPs from Plectranthus barbatus in Nicotiana benthamiana are efficient in boosting diterpenoid levels compared to coexpression of the Nicotiana endogenous DXS and GGPPs genes, which have no effect on boosting diterpenoid level. Pigment analysis of infiltrated leaves, overview	748238