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-MIBS进行深入的酶机制研究。这些生物催化剂也可用于酶促合成新的2-MIB类似物，通过系统地扩大其底物耐受性。将研究它们通过FPPS转化为GPP类似物，然后通过GPPMT和2-MIBS测试它们的转化。酶法制备的SAM衍生物也将用于进一步扩大可及的化学空间。在成功的情况下，产物将从制备规模温育中纯化，并且它们的结构将通过NMR确定。将开发基于发光的方法以有效地跟踪萜烯合酶活性。萜烯环化导致二磷酸作为偶联副产物，其可以通过硫酸腺苷酰转移酶与腺苷5 '-磷酸硫酸酯（APS）一起转化为ATP，然后通过萤火虫荧光素酶（如在焦磷酸测序中使用的）偶联至光产生反应。该装置将被优化，以允许容易地确定萜烯合酶的酶动力学，特别是2-MIBS。为了这个目的，需要确定酶的浓度，为此，将使用使用酶与绿色荧光蛋白融合的荧光方法。将对GPPMT和2-MIBS中高度保守的残基进行定点诱变，以研究这些酶的机理。对于活性酶变体，将通过荧光素酶系统测定动力学并与野生型活性进行比较。定向进化将用于扩大GPPMT和2-MIBS的活性位点空腔和底物耐受性，以使这些酶可用于更大的底物。优化的2-MIBS变体将用于产生具有反应性锚如炔丙基的2-MIB，其可用于点击化学。这使得可以通过接头将获得的2-MIB类似物连接到生物素，使整个分子固定在链霉抗生物素蛋白琼脂糖凝胶上，然后可以用于从细胞裂解物中捞出分子蛋白质靶点。这将用选定的细菌2-MIB生产者进行，以研究2-MIB是否在生产者本身中具有分子靶标。

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.