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.

重组Sm2OGD25的功能表征
摘要 丹参（Salvia miltiorrhiza）是最常用的中药材之一。丹参酮类是丹参中含量最丰富的脂溶性活性成分，属于一类结构高度氧化的松香烷型二萜，具备多种药理活性。尽管目前已有多种酶（包括二萜合酶、细胞色素P450及Fe(II)/2-氧戊二酸依赖型双加氧酶（2OGD））在松香烷型二萜的生物合成中得到功能表征，但丹参酮类高度氧化的结构与复杂的次生代谢网络提示，仍有更多氧化酶有待挖掘与表征。本研究从丹参中鉴定得到一种新型2OGD（Sm2OGD25）。体外分子克隆与功能研究表明，Sm2OGD25可分别催化苏铁杉酚（sugiol）的C-15和C-16位羟基化，分别生成海松烯酮B（hypargenin B）与克罗苏木素C（crossogumerin C）。DOXC家族系统发育分析显示，Sm2OGD25隶属于DOXC54进化支。进一步的结构建模与定点突变表征揭示了氢键结合残基Y339以及疏水残基（V122、F129、A144、A208、F303与L344）在底物结合与酶活性中的关键作用。本研究将推动植物2OGD的催化表征及二萜类次生代谢生物合成网络的后续研究。

方法 本研究采用超高效液相色谱-四极杆飞行时间质谱（UPLC-QTOF-MS，美国马萨诸塞州米尔福德市沃特世（Waters Technologies）公司）进行反应产物鉴定。样品于35 ℃下在ACQUITY UPLC® BEH C18色谱柱（2.1 mm × 100 mm，1.7 μm，美国沃特世公司）及配套ACQUITY UPLC® BEH C18保护柱上分离，酶解产物采用表S2所列线性梯度条件进行洗脱，进样体积为5 μL。转化率以百分比形式通过UPLC分析得到的产物与底物峰面积计算得到。飞行时间质谱检测采用配备电喷雾电离（ESI）负离子模式的Xevo G2-S QTOF（沃特世）系统完成，全扫描监测范围设为m/z 50–1500。其余操作参数如下：扫描时长0.1 s；检测时长18 min；锥孔电压40 V；高能碰撞电压梯度为20–50 V。数据采集与处理采用MassLynx 4.1版本软件（美国马萨诸塞州米尔福德市沃特世公司）完成。

使用说明 数据文件需使用MassLynx软件打开。