Functional characterization of recombinant Sm2OGD25

Functional characterization of recombinant Sm2OGD25. Abstract <em>Salvia miltiorrhiza</em> is one of the most commonly used Chinese medicinal herbs. Tanshinones, the most abundant lipo-soluble bioactive constituents of <em>S. miltiorrhiza</em>, are a class of structural highly oxidized abietane-type diterpenoids with multiple pharmacological activities. Although several enzymes including diterpene synthase, cytochrome P450, and Fe(II)/2-oxoglutarate-dependent dioxygenase (2OGD) have been functionally characterized in biosynthesis of abietane-type diterpenoids, the highly oxidized structure and complex secondary metabolic networks of tanshinones imply that more oxidases should be characterized. Here, we identified a new 2OGD (Sm2OGD25) from <em>S. miltiorrhiza</em>. Molecular cloning and functional studies <em>in vitro</em> showed that Sm2OGD25 could catalyze the hydroxylation of sugiol at C-15 and C-16 positions to produce hypargenin B and crossogumerin C, respectively. The phylogenetic analysis of the DOXC family demonstrated that Sm2OGD25 belongs to the DOXC54 clade. Furthermore, structural modeling and site-directed mutagenesis characterization revealed the importance of the hydrogen-bonding residue Y339 and the hydrophobic residues (V122, F129, A144, A208, F303 and L344) in substrate binding and enzyme activity. This work will promote further studies on the catalytic characterization of plant 2OGDs and the secondary metabolic biosynthesis network of diterpenoids. Methods Analyses were analyzed by UPLC-QTOF-MS (Waters Technologies, Milford, MA, USA) for the identification of reactions. Samples were separated on a ACQUITY UPLC® BEH C18 (2.1 mm × 100 mm, 1.7μm, Waters Technologies, USA) protected with a ACQUITY UPLC® BEH C18 guard column at 35 °C, and the enzymatic products were eluted with a linear gradient condition given in Table S2. 5 μL of sample was injected into the system. The conversion rates in percent were calculated from peak areas of products and substrates as analyzed by UPLC. Time-of-flight MS detection was performed with a Xevo G2-S QTOF (Waters) system equipped with electrospray ionization (ESI) operating in negative ion mode. Full scan monitoring range was performed in the range of <em>m/z</em> 50–1500. The other operating parameters were as follows: the scanning time of 0.1 s; the detection time of 18 min; and the cone voltage of 40 V; the high energy ramp collision voltage was 20–50 V. Data acquisition and processing were performed by MassLynx version 4.1 software (Waters Corp., Milford, MA, USA). <br> Usage Notes The data files should be open with Masslynx software.