Fe(II)/2-OG依赖型双加氧酶催化游离氨基酸选择性羟基化的分子机制及其功能调控

Fe(II)/2-OG-dependent dioxygenase has been widely used in synthesis of active pharmaceutical ingredients of various chemical structures, due to its catalytic diversity of reaction type. Establishment of the general reaction system involving synergistic interactions between the functional enzyme and necessary ligands including cofactors and co-substrates would be important for highly efficient synthesis of different pharmaceutical intermediates in an extensive way. Structure-based understanding the molecular mechanism of selective recognition and interaction between Fe(II)/2-OG-dependent dioxygenase and the ligands and even function regulation of the enzyme towards target reactions would be the key issues for building the general catalytic system of Fe(II)/2-OG-dependent dioxygenase. Concerning Fe(II)/2-OG-dependent dioxygenase catalyzing selective hydroxylation of free amino acids, this research is to discover new enzymes by the approach combining bioinformatics and molecular modelling to mine sequence databases, to solve the molecular mechanism of Fe(II)/2-OG-dependent dioxygenase binding the substrates of free amino acids and catalyzing their hydroxylation selectively, and to enhance the availability of newly discovered Fe(II)/2-OG-dependent dioxygenase for industrial application by redesigning of the “hot spots” of active sites involved in ligand binding and molecular interactions. This research would provide the basic theory and research foundation for development of the general technology platform of highly efficient biosynthesis of active pharmaceutical ingredients of hydroxylated derivatives.

Fe(II)/2-OG依赖型双加氧酶的反应多样性使其广泛用于不同化学结构医药中间体的合成，建立该类酶与反应必需辅助因子和共底物协同作用的共性反应体系，对于重要医药中间体的广谱性高效生物合成具有重要意义。如何基于结构认知探究Fe(II)/2-OG依赖型双加氧酶与反应必需配体分子之间的选择性识别和作用机制，并针对目标反应调控强化功能酶的催化性能，是基于Fe(II)/2-OG依赖型双加氧酶建立共性反应的关键科学问题。本项目拟针对Fe(II)/2-OG依赖型双加氧酶催化游离氨基酸选择性羟基化反应，采用新型生物信息学和分子模拟相结合的功能酶基因探矿方法，挖掘新型功能酶；基于底物广谱性表征，解析Fe(II)/2-OG依赖型双加氧酶选择性识别游离氨基酸及催化位置选择性羟基化的分子机制；基于热点设计策略，拓展和强化催化性能，为生物合成羟基化衍生类重要医药中间体广谱开发共性技术体系的建立提供理论和实践基础。

Fe(II)/2-OG依赖型双加氧酶的反应多样性使其广泛用于不同化学结构医药中间体的合成，建立该类酶与反应必需辅助因子和共底物协同作用的共性反应体系，对于重要医药中间体的广谱性高效生物合成具有重要意义。本项目采用结构功能相关序列和结构分子模拟相结合的功能酶基因挖掘方法，在基因（组）数据库中搜索潜在新酶序列信息，获得选择性羟基化游离氨基酸的新型Fe(II)/2-OG 依赖型双加氧酶；基于基因挖掘获得的新型Fe(II)/2-OG 依赖型双加氧酶和已有Fe(II)/2-OG 依赖型双加氧酶IDO的结构信息，在分子水平探究其催化表征的分子机制，进一步建立了重组菌全细胞催化转化羟基异亮氨酸的体系与过程调控规律；优化Fe(II)/2-OG 依赖型双加氧酶IDO、KaPH1等在重组系统中的表达水平，构建双加氧酶表达和催化转化耦联的体系和过程，进一步构建体外多酶催化级联反应体系，实现双加氧酶催化必需辅因子2-酮戊二酸的耦联再生，从而提升Fe(II)/2-OG 依赖型双加氧酶催化游离氨基酸羟基化的反应效率。相关研究成果发表论文17篇，其中SCI论文16篇、CSCD论文1篇；申请中国发明专利1项，授权中国发明专利2项；获得中国石油和化学工业联合会技术发明奖一等奖1项、中国石油和化学工业联合会青年科技突出贡献奖1项。研究结果为生物合成羟基化衍生类重要医药中间体广谱开发共性技术体系的建立提供理论和实践基础。