EAPSI: Crystallization of Mutant N-demethylase Enzymes

EAPSI：突变型 N-去甲基酶的结晶

• 批准号: 1713935
• 负责人: Shelby Brooks
• 金额: $ 0.54万
• 依托单位: Brooks Shelby G
• 依托单位国家: 美国
• 项目类别: Fellowship Award
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-06-01 至 2018-05-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1713935&HistoricalAwards=false
• 关键词: EAPSI; Crystallization; Mutant; demethylase; Enzymes

The goal of this research project is to determine the crystal structures of mutant enzymes derived from the bacterial caffeine-degrading enzymes NdmABCDE. These enzymes are used to sequentially remove the N1-, N3, and N7-methyl groups from the caffeine molecule. This N-demethylation process can result in a new, economical, and environmentally-friendly method to produce valuable methylxanthies from caffeine. NdmA and NdmB perform the first two steps by removing the N1-methyl group followed by the N3-methyl group, respectively. NdmCDE form a large protein complex to carry out the N7-demethylation of 7-methylxanthine to xanthine. We have created several mutant N¬-demethylase enzymes with improved abilities to produce high-value metabolites from caffeine, but the yields of these metabolites are still low. Crystal structures of the mutant caffeine-degrading enzymes with the metabolites they degrade will be determined under the mentorship of Dr. Hyun Kyu Song of Korea University. The crystal structures will provide insight into rationally designing new mutant enzymes to increase the yield of high-value biochemicals from this process. Dr. Song's research group is the only one to determine the structure of the N-demethylase enzymes to date. The Song lab, thus, has all of the protocols and equipment necessary to complete this project. The N-demethylation of caffeine (1,3,7-trimethylxanthine) can provide valuable methylxanthines in an economical, environmentally-friendly method compared to the current chemical synthesis. Paraxanthine (1,7-dimethylxanthine) is produced as a minor product through the N3-demethylation of caffeine due to slight promiscuity of the N1-demethylase NdmA toward the N3-methyl group. The N3-demethylase NdmB catalyzes the conversion of theobromine (3,7-dimethylxanthine) to 7-methylxanthine, but exhibits very low activity toward molecules that contain an N1-methyl group. Mutants of NdmA and NdmB have been created to better understand the substrate interactions with the enzymes. The hypothesis of this work is that the mutation of two residues in the NdmA active site to those found at similar locations in NdmB will swap the NdmA activity from N1-demethylation to N3-demethylation, resulting in higher yields of paraxanthine. Under the direction of Dr. Song, the researcher will determine the crystal structures of these mutants with their substrates and discover the substrate orientation in the enzyme to ultimately optimize the yield of paraxanthine. Additionally, the project will also seek to determine the crystal structure of the native NdmCDE complex. The knowledge gained through these crystallographic studies will enable rational engineering of bacterial N-demethylase enzymes and accelerate the biosynthesis of high-value methylxanthines for applications in the pharmaceutical and cosmetic industries. This award, under the East Asia and Pacific Summer Institutes program, supports summer research by a U.S. graduate student and is jointly funded by NSF and the National Research Foundation of Korea.

该研究项目的目标是确定来自细菌咖啡因降解酶NdmABCDE的突变酶的晶体结构。这些酶用于依次从咖啡因分子中去除N1-、N3和N7-甲基。这种N-脱甲基化过程可以产生一种新的，经济的，环境友好的方法来从咖啡因生产有价值的甲基黄嘌呤。NdmA和NdmB分别通过去除N1-甲基和N3-甲基进行前两步。NdmCDE形成大的蛋白质复合物以进行7-甲基黄嘌呤的N7-去甲基化为黄嘌呤。我们已经创造了几种突变的N-脱甲基酶，其具有改进的从咖啡因产生高价值代谢物的能力，但是这些代谢物的产量仍然很低。突变咖啡因降解酶及其降解代谢物的晶体结构将在韩国大学Hyun Kyu Song博士的指导下确定。晶体结构将为合理设计新的突变酶提供洞察力，以提高该过程中高价值生化品的产量。宋博士的研究小组是迄今为止唯一确定N-脱甲基酶结构的研究小组。因此，Song实验室拥有完成该项目所需的所有协议和设备。与目前的化学合成相比，咖啡因的N-去甲基化（1，3，7-三甲基黄嘌呤）可以以经济、环境友好的方法提供有价值的甲基黄嘌呤。副黄嘌呤（1，7-二甲基黄嘌呤）是通过咖啡因的N3-去甲基化产生的次要产物，这是由于N1-去甲基酶NdmA对N3-甲基的轻微混杂。N3-脱甲基酶NdmB催化可可碱（3，7-二甲基黄嘌呤）转化为7-甲基黄嘌呤，但对含有N1-甲基的分子表现出非常低的活性。已经创建了NdmA和NdmB的突变体，以更好地理解底物与酶的相互作用。这项工作的假设是，NdmA活性位点中的两个残基突变为NdmB中相似位置处的那些残基，将NdmA活性从N1-去甲基化交换为N3-去甲基化，从而导致副黄嘌呤的产率更高。在宋博士的指导下，研究人员将确定这些突变体与其底物的晶体结构，并发现酶中的底物方向，以最终优化副黄嘌呤的产量。此外，该项目还将寻求确定天然NdmCDE复合物的晶体结构。通过这些晶体学研究获得的知识将使细菌N-脱甲基酶的合理工程化，并加速高价值甲基黄嘌呤的生物合成，用于制药和化妆品行业。该奖项是东亚和太平洋夏季研究所计划下的一个奖项，支持美国研究生的夏季研究，由NSF和韩国国家研究基金会共同资助。