The research group led by Dr. Xiaoqiang Huang at the School of Chemistry and Chemical Engineering, Nanjing University, has engineered thiamine pyrophosphate-dependent enzymes and developed a photobiocatalysis system, unlocking a new-to-nature three-component radical biocatalysis. This work, titled Synergistic photobiocatalysis for enantioselective triple radical sorting, was published online in Nature on November 22, 2024. Link to the paper:
Notably, in December 2023, Huang’s group published their first paper in this field in Nature. Please refer to
Enzymes are nature’s catalysts, playing crucial roles in various physiological processes of life systems and serving as important tools in fundamental research and biomanufacturing. However, the catalytic functions of natural enzymes are relatively limited and often fall short of current demands. For instance, the multicomponent synthesis of complex compounds in nature often requires the cooperative catalysis of multiple enzymes. Although significant advancements have been made in enzyme catalysis in recent years, most enzyme-catalyzed reactions currently developed involve single or bi-molecular transformations. The inhibition of naturally selected enzymes’ inherent reactivity and the use of a single protein to orchestrate the ordered transformation of three different substrates/chemical intermediates remain significant challenges.
Addressing these issues, Dr. Huang Xiaoqiang’s team utilized visible light excitation and protein engineering to “reshape” the benzaldehyde lyase into a three-component radical enzyme (3CRE), achieving a non-natural high enantioselective three-component radical coupling reaction. They also elucidated the mechanism of this new function through mechanistic experiments. This system can combine three variable substrates, greatly enriching the diversity of photobiocatalysis and marking a significant achievement in the field (Figure 1).
Figure 1：Milestones in the field of photobiocatalysis
The team selected 4-phenylbenzaldehyde (1a), styrene (2a), and bromoacetone (3a) as model substrates to explore the dual catalytic system consisting [Ru(bpy)3]Cl2?6H2O and a ThDP-dependent enzyme. Through molecular dynamics simulations and a semi-rational iterative site-specific mutagenesis strategy, they engineered the enzyme. The results showed that the T481L and A480G mutations significantly improved the enantioselectivity of the reaction, while the Q113H and N283F mutations notably enhanced the reaction yield. Finally, an optimal variant was obtained after five rounds of mutagenesis (Figure 2).
Figure 2：The development of three-component photobiocatalysis
The photobiocatalysis exhibited excellent substrate tolerance, accepting a wide range of substituted aromatic aldehydes, heterocyclic aromatic aldehydes, various substituted aryl olefins, even alkyl olefins, as well as multiple alkyl radical precursors (Figure 3). The study presented 33 examples, of which 25 achieve ≥97% enantiomeric excess (ee), highlighting the power of the enzyme’s tunable active site in controlling the stereochemistry of free radicals.
Figure 3：Scope investigation.
Huang’s group, in collaboration with Wang’s lab at Xiamen University, conducted a detailed study of the mechanism using a combination of wet experiments and theoretical calculations. They revealed the key factors that enable the three-component reactivity and discussed the origin of its high stereochemical selectivity. For further details, please refer to the original paper.
This work was supported by National Natural Science Foundation projects (22277053, 22122305, 21927814, 223B2703), National Key Research and Development Program projects (2022YFA0913000, 2019YFA0405600), Natural Science Foundation projects of Jiangsu Province (BK20220760), the Central University Basic Research Fund Project (0205/14380346), Nanjing University Excellence Program Project (ZYJH004), and Chinese Academy of Sciences Strategic Key Research Project (XDB0540200).
