立体异构酶MarH和StnK3的结构及其催化机制的研究

β-methyl amino acids are important building blocks of many bioactive natural products. (2S, 3S)- and (2S, 3R)-β-methyl-tryptophan (β-MeTrp) are the important biosynthetic precursors of Maremycins A/B (MARs) and Streptonigrin (STN), respectively. It was found that MarH, the stereoisomerase involved in the biosynthesis of MARs, and StnK3 from the biosynthetic gene cluster of STN catalyze the configurational inversion of (R)-β-methyl-indolepyruvate (β-MeInPy) to form (S)-β-MeInPy, which is the key biosynthetic step to determine whether (2S, 3R)- or (2S, 3S)-β-MeTrp will be further formed. However, the catalytic mechanism of MarH or StnK3 is still not clear so far. . To better understand their catalytic mechanism, it is proposed to determine the structures of MarH and StnK3 and their complexes with the substrate or the substrate analogues by solution NMR. We successfully expressed the 15N labelled MarH and StnK3 and obtained their 15N HSQC spectra. The aromatic rings may involve in the interaction between the enzymes and their substrates, which are of importance to illustrate the catalytic mechanism of MarH and StnK3, and also to reveal their selectivity on the specific substrate at the structural level. Accordingly, 3D (Hali)CaliCaroHaro-TOCSY experiment is specifically developed for the chemical shift assignment of the aromatic rings, which will also be of great help to improve the resolution of the obtained NMR structures. Moreover, using gene engineering techniques, targeted modification of the catalytic function of MarH or StnK3 is further explored, aiming to achieve controlled regulation of their selectivity on the substrate so as to make them to obtain catalytic activity on some non-native substrates and to broaden the scope of substrates applicable to them, ultimately to be able to apply the gene-engineered stereoisomerases in the green synthesis.

β-甲基氨基酸是许多活性天然产物的构成单元。(2S,3S)-和(2S,3R)-两种立体结构不同的β-甲基色氨酸分别是链霉菌素（MARs）和链黑菌素（STN）的生物合成前体。MARs和STN生物合成酶簇中的立体异构酶MarH和StnK3能催化(R)-β-甲基吲哚丙酮酸结构反转成S构型，是决定进一步形成(2S,3R)-或(2S,3S)-β-甲基色氨酸的关键步骤。但目前对它们的催化机制并不清楚。.本项目拟通过液态核磁共振技术确定MarH和StnK3及它们与底物复合物的三维结构，以阐明它们的催化机制。我们成功地表达了两种酶并获得HSQC图谱。由于芳香环可能参与酶与底物间的结合与催化作用，本项目将发展(Hali)CaliCaroHaro-TOCSY核磁方法用于芳香环信号的归属，并将探索通过基因改造调控两种酶对底物的选择性，使其获得对某些非天然底物的催化性，拓宽底物范围，寻求异构酶在绿色合成中的应用。

项目背景：.β-甲基氨基酸是许多活性天然产物的构成单元。(2S,3S)-和(2S,3R)-两种立体结构不同的β-甲基色氨酸分别是链霉菌素（MARs）和链黑菌素（STN）的生物合成前体。MARs和STN生物合成酶簇中的MarH和StnK3能催化(R)-β-甲基吲哚丙酮酸结构反转成S构型，是决定进一步形成(2S,3R)-或(2S,3S)-β-甲基色氨酸的关键步骤。但目前对它们的催化机制并不清楚。.主要研究内容：.研究了MarH的三维结构，以及MarH与底物类似物L-Trp形成的复合物结构，基于三维结构分析，发现并进一步通过突变验证了MarH催化关键氨基酸，解释MarH催化异构化的机理。优化表达和纯化了Stnk3。.重要结果：.解析了MarH的晶体结构。基于MarH晶体结构，结合核磁共振数据分析确定了MarH在结合底物类似物L-Trp前后结构基本不变。在此基础上，结合晶体结构及核磁共振filtered-NOE图谱包含的分子间（MarH和底物类似物L-Trp之间）距离约束信息，使用Xplor-NIH计算得到MarH与底物类似物L-Trp的复合物结构。通过三维结构分析，确定了底物类似物L-Trp与MarH催化口袋中残基间的相互作用，并确认了作为催化质子受体的氨基酸His25。通过突变失活，进一步验证了His25在MarH催化过程中的关键作用。在底物反应位点有效距离范围内，未能发现可以作为质子供体的氨基酸，从而提出水分子作为质子供体参与催化反应，最终阐明了MarH的催化机制。成功表达了立体异构酶Stnk3。.关键数据：.通过基于HSQC图谱作为参考的样品优化制备得到可用于结构解析的MarH蛋白样品，首先采集游离态MarH（无底物）和复合态MarH（与底物类似物L-Trp所形成复合物）的一系列核磁共振图谱数据，并分别对两种状态下MarH进行了主、侧链归属，基于归属的化学位移利用TALOS软件预测得到了二级结构信息。利用晶体衍射技术解析了构建优化后的游离态MarH在三种溶液条件下的晶体结构。基于游离态MarH的晶体结构以及MarH与底物类似物L-Trp所形成的复合物的核磁共振filtered-NOE图谱，转换得到的距离约束，计算得到MarH与L-Trp的复合物结构。.科学意义：.发现了MarH催化异构化反应过程中关键的催化氨基酸，解释了其催化机制。为进一步拓展底物范围的定向酶功能改