# Cloning and Characterization of Two Iridoid Synthase Homologs from _Swertia Mussotii_
## 2 Results
### Functional Characterization and Substrate Preferences of Recombinant SmIS1 and SmIS2  
The full-length ORF of _SmIS1_ cDNA was subcloned into pETMALc-H. This vector was used to express a fusion protein with a maltose-binding protein at the N-terminus. The full-length ORF of SmIS2 cDNA was subcloned into pET-28a to produce a fusion protein with 6-His tags at both the _N_-terminus and C-terminus. Regarding SmIS1 heteroexpression, we also subcloned the SmIS1 ORF cDNA into the pET-28a vector to produce a fusion protein with His tags, which is a smaller tag. However, the SmIS1 recombinant protein always accumulated in inclusion bodies. Recombinant  
Figure 4: Phylogenetic tree of SmIS1 and SmIS2 together with 34 known P5 bRs. The phylogenetic tree was drawn using _P. patens_ (GenBank: EDQ81106.1) as an outgroup and the following amino acid sequences with accession IDs noted in brackets: _C. roseus_ P5 bR1 (AIW09143.1); _C. roseus_ P5 bR3(AIW09145.1); _Nerium oleander_ P5 bR-B (ADC56540.1); _C. roseus_ P5 bR2 (AIW09144.1); _Gomplocarpus fruticosus_ P5 bR (ADG56546.1); _Asclepius curassavica_ P5 bR (ADG56538.1); _Withania somnifera_ P5 bR (AEV82379.1); _Nicotiana tabacum_ P5 bR (BAH47641.1); _Duboisia hopwoodii_ P5 bR (AFZ41795.1); _Genlisea aurea_ P5 bR (EPS65468.1); _Mentha x piperita_ P5 bR (ADG46022.1); _Digitalis lanata_ P5 bR1(AAS93804.1); _Digitalis purpurea_ P5 bR1(AAS93805.1); _Vitis vinifera_ P5 bR (ALB78111.1); _Thlaspi densiforum_ P5 bR (ALD83449.1); _Arabidopsis thaliana_ At4g24220 (NP_001078438.1); _Medicago truncatula_ P5 bR1 (AIW09149.1); _Medicago truncatula_ P5 bR2 (AIW09150.1); _Passiflora incarnata_ P5 bR (AFW16644.1); _C. roseus_ P5 bR 4 (AIW09146.1); _Ola europaea_ 1,4-R 1.1B (ALV83440.1); _Olea europaea_ 1,4-R 1.2B (ALV83440.1); _Olea europaea_ 1,4-R 1.2A (ALV83441.1); _Camptothea acumnata_ CaIS (AON76722.1); _Lonicera japonica_ LiJS (AMB61018.1); _C. roseus_ CrIS (AFW98981.1); _Olea europaea_ Oels (ALV83438.1); _D. purpurea_ P5 bR2 (ACZ66261.1); _D. lanta_ P5 bR2 (ADL28122.1); _C. roseus_ P5 bR6 (AIW09148.1); _Medicago truncatula_ P5 bR3 (AIW09151.1); _Medicago truncatula_ P5 bR3 (AIW09152.1). enzymes were produced in sufficient amounts to allow further characterization of SmIS1 and SmIS2. Affinity purification of both recombinant proteins resulted in single bands of the expected sizes that were visible on Coomassieie-stained SDS-PAGE gels. A \(\sim\)88 kDa protein band was observed in the SmIS1 lane (including 10 histidines and a MBP protein), and a \(\sim\)46 kDa protein was observed in the SmIS2 lane (including two 6 histidines) (Figure 5). These findings indicated that SmIS1 and SmIS2 were successfully expressed in _E. coli_ cells. To determine whether recombinant SmIS1 and SmIS2 were both catalytically active, as we predicted, an in vitro assay to determine their enzyme activities and to detect their products was performed. We used 8-oxogeranial as a substrate for the enzyme assays in GC-MS. The enzymes were incubated with substrates in the presence of NADPH. Based on their mass, elemental composition, and fragmentation pattern and the enzyme activity of other known IS [13; 15], the products were characterized as neepetalactol and iridodials (Figure 6: peak 3, peak 4 and peak 5). The substrate was a mixture of 8-oxoneral (peak 1) and 8-oxogeranial (peak 2). SmIS1 and SmIS2 turned over both substrates well (Figure 6) [13]. Figure 5: Heterologous expression of SmIS1 and Smis2 cDNA in _E. coli._ Lane SmIS1, purified recombinant SmIS1; lane SmIS2, purified recombinant SmIS2. Figure 6: Functional characterization of SmIS1 and Smis2. (**A**) Analysis of the reaction product by GC-MS. Control, the reaction in the presence of denatured SmIS1 or SmIS2 proteins; (**B**) MS spectra of the substrates (peaks 1 and 2) and products (peaks 3, 4, and 5). The steady-state kinetic constants of the reactions were determined using spectrophotometric NADPH consumption assays (Table 1, Figure S1).