A natural enzyme that catalyzes competitive 2+2 and 4+2 cycloaddition reactions

On January 23, 2023, Prof. Wen Liu and Prof. Lifeng Pan from the Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, and Ken Wen, Prof. Li Pan, Department of Chemistry, University of California, Los Angeles (UCLA), and N. Houk’s lab collaborated to publish a study titled “A cyclase that catalyses competing 2+2 and 4+2 cycloadditions” in the journal Nature Chemistry.

This research work discovered a novel cycloaddition enzyme that can catalyze the competitive 2+2 and 4+2 cycloaddition reactions, and precisely regulated its catalytic selectivity based on computationally assisted directed evolution design, which laid an important foundation for the development and industrial application of subsequent cycloaddition enzymes.

The first authors of the paper are Wang Hongbo, Zou Yike, Li Miao and Tang Zhijun, and the corresponding authors are Liu Wen, Pan Lifeng and Ken. N. Houk。

Cycloaddition reaction is one of the most widely used reactions in chemical synthesis, which can effectively realize the chiral construction of heterocyclics, spirocycles and bridge rings, and its mechanism research and result prediction are important achievements of Woodward-Hoffmann rule and frontier orbital theory. Chemists have developed a variety of metal and small molecule catalysts for catalyzing cycloaddition reactions and their regional and stereoselective regulation. Nature implements these cyclization reactions through various enzymes, including Diels-Alderases, which catalyze synergistic 4+2 cyclization, but biosynthetic enzymes with 2+2 cyclase activity have not been discovered.

In the previous research work, Liu Wen’s group discovered a two-step complete enzyme-dependent synergistic 4+2 cyclization reaction, responsible for the formation of chiral naphthalene rings and spirocyclic rings, which was used for the construction of the five-ring rigid skeleton of pyrroindolycin, which provided strong evidence for confirming the natural existence of Diels-Alderase (J. Am. Chem. Soc., 2012, 134, 17342-17345;Nat. Chem. Biol., 2015, 11, 259-265)。 Liu Wen’s research group and Pan Lifeng’s research group and K. K. N. Houk’s group collaborated to explore and verify the co-occurrence mechanism of the corresponding enzymatic reaction based on crystal structure and chemical calculations (Cell Chem. Biol., 2016, 23, 352-360; Cell. Chem. Biol. 2018, 25, 718-727;J. Am. Chem. Soc. 2020, 142 , 20232-20239;J. Am. Chem. Soc. 2022, 144 , 5099-5107)。

In this study, the authors discovered a novel cycloaddition enzyme responsible for chiral spirocyclic formation in the biosynthetic pathway of pyrrosporine, a structural analogue of pyrroindolmycin. The enzyme is a class of β-barrel-folded cyclases that do not depend on the activation of light, and when the intermediate of romidomycin is used as the substrate, in addition to exo- and endo-4+2 addition products, exo-2+2 addition products are also produced. Using structural biology and computational chemistry, the authors analyzed the molecular mechanism of the enzyme to stimulate different cycloaddition reactions and achieve regional and stereoselective regulation, and derived and verified the reaction process of generating thermodynamically favorable 4+2 and kinetically favorable 2+2 products through double radical intermediates. Through directed evolution, the authors precisely regulated the chemical and stereoselectivity of the enzyme, and obtained mutant proteins that catalyze the single cycloaddition reaction of exo-4+2, endo-4+2 and exo-2+2, respectively.

Figure 1: Competitive 2+2/4+2 cycloaddition reactions catalyzed by β-barrel proteins and directed evolution based on selective regulation

This study reports a rare non-photoactivated enzymatic 2+2 cycloaddition reaction, providing a paradigm for improving catalytic selectivity and enzymatic activity through the process of enzyme-substrate co-evolution. This work is supported by the Ministry of Science and Technology, the National Natural Science Foundation of China, the Chinese Academy of Sciences, and the Shanghai Municipal Science and Technology Commission. (Source: Science Network)

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