Investigations on the Biosynthetic Enzymes for the Earthy Odorant 2-Methylisoborneol

本项目旨在对2-MIB生物合成酶GPPMT和2-MIB的酶机理进行深入的研究。这些生物催化剂还可用于酶法合成新的2-MIB类似物，首先，通过化学合成得到几个DMAPP和IPP类似物。将研究它们通过FPPS转换为GPP类似物，然后通过GPPMT和2-MIB测试它们的转换。此外，还将使用酶法制备的SAM衍生物来进一步扩大可接近的化学空间。在成功的案例中，产物将从制备规模的孵化中提纯，其结构将通过NMR确定。将开发一种基于发光的方法来有效跟踪萜烯合成酶的活性。萜类环化反应产生偶联副产物二磷酸，该副产物可与5‘-磷酸腺苷(APS)通过硫酸酯基腺苷转移酶转化为ATP，然后偶联到萤火虫荧光素酶的发光反应中(如焦化测序中所用)。该装置将进行优化，以便轻松地测定萜烯合酶动力学，特别是2-MIBs。为此，需要确定酶的浓度，为此将使用酶融合到绿色荧光蛋白的荧光法。将对GPPMT和2-MIBs中高度保守的残基进行定点突变，以从机理上研究这些酶。对于活性酶变异体，动力学将由荧光素酶系统确定，并与野生型活性进行比较。定向进化将用于扩大GPPMT和2-MIB的活性部位空穴和底物耐受性，使这些酶可用于更大的底物。优化的2-MIBs变体将用于生产具有反应性锚基的2-MIB，例如可用于点击化学的炔丙基。这使得通过连接物将获得的2-MIB类似物连接到生物素成为可能，使整个分子在链霉亲和素琼脂糖凝胶上固定，然后可以用来从细胞裂解产物中筛选出分子蛋白质靶标。这将在选定的细菌2-MIB生产者中完成，以调查2-MIB生产者本身是否有分子靶标。

This project aims at a deep enzyme mechanistic investigation of the 2-MIB biosynthetic enzymes GPPMT and 2-MIBS. These biocatalysts shall also be made available for the enzymatic synthesis of novel 2-MIB analogs by expanding their substrate tolerance systematically.First, several DMAPP and IPP analogs will be obtained by chemical synthesis. Their conversion into GPP analogs by FPPS will be investigated, followed by testing their conversion by GPPMT and then 2-MIBS. Also enzymatically prepared SAM derivatives will be used to further expand the accessible chemical space. In successful cases the products will be purified from preparative scale incubations and their structures will be determined by NMR.A luminescence based method will be developed to efficiently follow terpene synthase activity. Terpene cyclizations lead to diphosphate as a coupled byproduct that can be converted with adenosine 5’-phosphosulfate (APS) by sulfate adenylyltransferase into ATP, followed by coupling to the light generating reaction by firefly luciferase (as used in pyrosequencing). The setup will be optimized to allow for the easy determination of terpene synthase enzyme kinetics, especially of 2-MIBS. For this purpose enzyme concentrations need to be determined for which a fluorescence method using enzyme fusions to the green fluorescent protein will be used.Site-directed mutagenesis of highly conserved residues in GPPMT and 2-MIBS will be performed to study these enzymes mechanistically. For active enzyme variants kinetics will be determined by the luciferase system and compared to wildtype activity. Directed evolution will be used to expand the active site cavities and substrate tolerances of GPPMT and 2-MIBS to make these enzymes accessible for larger substrates. An optimized 2-MIBS variant will be used to produce 2-MIB with a reactive anchor such as a propargyl group that can be used in click chemistry. This makes it possible to connect the obtained 2-MIB analog via a linker to biotin, making the whole molecule immobilizable on streptavidin sepharose, which can then be used to fish out the molecular protein targets from cell lysates. This will be done with selected bacterial 2-MIB producers to investigate if 2-MIB has a molecular target in the producers themselves.