# Cloning and Characterization of Two Iridoid Synthase Homologs from _Swertia Mussotii_
## 2 Results  
### Cloning, Sequencing and Phylogenetic Analysis of SmIS1 and SmIS2 cDNAs  
The SmIS1 and SmIS2 gene sequences were retrieved from whole transcriptome data for _S. mussotii_ using a BLAST similarity search program ([[http://www.ncbi.nlm.nih.gov/BLAST](http://www.ncbi.nlm.nih.gov/BLAST)]([http://www.ncbi.nlm.nih.gov/BLAST](http://www.ncbi.nlm.nih.gov/BLAST))). The cDNA was cloned and its sequence was analysed using total RNA isolated from young leaves of the _S. mussotii_ plant. The cDNA was used as a template for PCR, which was performed using primers designed for conserved regions in the 5\({}^{\prime}\) and 3\({}^{\prime}\) ends of the open reading frame (ORF). The sequences were submitted to GenBank and assigned accession numbers MF044036 (SmIS1) and MF044037 (SmIS2). The putative SmIS1 cDNA contained an ORF of 1173 bp that encoded a protein with 390 amino acid residues, and the putative SmIS2 cDNA contained an ORF of 1170 bp that encoded a protein with 389 amino acid residues. The calculated molecular mass and estimated pI of SmIS1 were 44.2 kDa and 6.08, respectively. The calculated molecular mass and estimated pI of SmIS2 were 43.9 kDa and 5.60, respectively. To calculate the amino acid sequence identity of the two proteins and other orthologues, multiple alignments were performed. SmIS1 and SmIS2 sequences were highly similar to those of CrIS and OeIS. SmIS1 shared 76.47% and 70.05% identity with CrIS and OeIS, respectively. SmIS2 shared 58.26% and 58.29% identity with CrIS and OeIS, respectively. The sequence analysis confirmed that SmIS1 and SmIS2 contained six conserved short-chain dehydrogenase/reductase (SDR) motifs and that these proteins therefore belong to the P5\(\beta\)Rs group [16], which also includes CrIS and OeIS. Tyr180 (178) and Lys178 (146) were observed in the conserved motifs of SmIS1 and SmIS2, respectively. These residues may control the enzymatic activity and substrate specificity of the proteins (Figure 2). The 3D structures of the proteins were modelled based on plant P5\(\beta\)Rs crystal structures that were available in the Protein Database ([[http://www.rcsb.org/pdb/home/home.do](http://www.rcsb.org/pdb/home/home.do)]([http://www.rcsb.org/pdb/home/home.do](http://www.rcsb.org/pdb/home/home.do))). Analyses of their entire lengths revealed that SmIS1 and SmIS2 were 57-78% identical to _D. lanta_ 2V6G and _C. roseus_ 5 COB, which have solved protein structures. Using _C. roseus_ CrIS (PDB: 5 COB) as a template for homology modelling, we generated 3D models of SmIS1 and SmIS2 (Figure 3). The SmIS1 model was 36.15% a-helices, 10.26% \(\beta\)-strands, and 53.59% loop structures. The SmIS2 model was 35.99% a-helices, 11.31% \(\beta\)-strands, and 52.70% loop structures. Furthermore, the GMQE scores of the modelled SmIS1 and SmIS2 were 0.89 and 0.80, respectively, and their QMEAN Z-Scores were \(-\)1.29 and \(-\)1.88, respectively. These data confirm that the structural models are robust and potentially useful for generating hypotheses about their substrates. We performed a phylogenetic analysis using the amino acid sequences of SmIS1 and SmIS2 and their homologues in other plants (Figure 4). All of the compared species belonged to the P5\(\beta\)Rs  
Figure 3: Predicted three-dimensional homology structure of SmIS1 (**A**) and SmIS2 (**B**) using recombinant CrIS from _C. roseus_ (SCOB) as a template. Helices and sheets are represented in red and blue, respectively. Figure 2: Amino acid sequence comparison of SmIS1 and Smis2 with other ISs. CrIS (_C. roseus_, GenBank: AFW98981.1); Oels (_O. europaea_, GenBank: ALV83438.1). The conserved motifs of the P5\(\beta\)Rs are indicated with bars. The highly conserved residues are highlighted in black, whereas similar residues are highlighted in grey. group. SmIS2 belonged to one subclade that clustered with _C. roseus_ P5 bR4 (AIW09146.1), _O. europaea_ 1,4-R1.1B (ALV83440.1), _O. europaea_ 1,4-R1.2B (ALV83442.1), _O. europaea_ 1,4-R1.2A (ALV83441.1), and _O. europaea_ 1,4-R1.1A (ALV83439.1).