On September 22, the reporter learned from Huazhong University of Science and Technology that a research paper by the team of Professors Fangrui Zhong and Yu Zhou Wu from the School of Chemistry and Chemical Engineering and Professor Xi Chen from Northwestern University was published in Nature. The research originally proposed a new concept of "triple-state photoenzyme", and the team developed a new class of artificial enzyme biocatalysts through the frontier technology of synthetic biology, which combines the advantages of unnatural reactivity of chemical synthesis and the precision and efficiency of biosynthesis, providing a new theory and technology for the green biomanufacturing of important functional chemicals in the fields of medicine and materials. The research results will contribute to the green biomanufacturing and the development of new technologies.

This research result will help green biomanufacturing and promote the evolution of traditional chemical chemical production to biochemical production.

Enzymes, as highly efficient biocatalysts formed by the long evolution of nature, are often only applicable to specialized substrates and natural chemical reactions of life, which can hardly meet the demand for the synthesis of diverse functional chemicals in social production.

In recent years, with the development of synthetic biology theory and technology, artificial enzymes can be constructed by artificially designing and introducing unnatural active centers in proteins, which can greatly expand the catalytic reactivity of enzymes and thus achieve more diverse biocatalytic synthesis of unnatural organic chemicals. While the number and variety of biological enzymes formed by natural evolution are numerous and functionally diverse, the vast majority are based on thermochemically driven activation mechanisms.

"Natural photoenzymes that can be driven by light are scarce, and only a few have been identified, including photosynthesis-related protochlorophyllate reductase, photo lyase for repairing DNA damage, and fatty acid decarboxylase." The above research team introduced that the fusion of photocatalysts and photocatalytic mechanisms developed by synthetic chemistry into proteins to construct new artificial photolases can break through the limitations of thermal catalytic mechanisms of natural enzymes, fundamentally expand the types of biocatalytic reactions, and provide new theories and technologies for green biomanufacturing of important functional chemicals.

Meanwhile, chirality is closely related to life phenomena and significantly affects material properties. The precise synthesis of chiral molecules can provide core technological support for the development of pharmaceutical, pesticide, information and material fields. Asymmetric catalysis is an efficient way to prepare chiral molecules, and tremendous progress has been made in this field in the past half century. However, the chiral control of photochemical reactions in the excited state is still a great challenge.

To address these problems, the research of Fangrui Zhong and Yuzhou Wu's team provides an original solution for the chiral catalytic synthesis of excited-state photoreactions. Based on the interdisciplinary background of organic synthesis, genetic engineering, protein engineering, enzyme theoretical computation and structural biology, the team inserted benzophenone-like superior photosensitizers developed by synthetic chemistry into the chiral cavity of selected proteins through gene codon expansion technology to construct an artificial photoenzyme TPe containing unnatural catalytic active centers.

From the first generation triplet-state photolase TPe1.0 constructed by chemical modification, the research team optimized the amino acid residues and reaction cavity structure of the enzyme by four rounds of mutation iterations, established a mutant library, completed the directed evolution of the photolase, and finally obtained the excellent mutant TPe4.0.

The single-crystal structure of the photoenzyme and substrate complexes was resolved by X-Ray by Chen's team, and the excellent chiral selectivity of the reaction was elucidated from the synergistic effect of multiple weak bonds such as hydrogen bonds formed between the photosensitizer and the surrounding key amino acid residues and the substrate.

This study shows that by combining the photocatalytic mechanism of triplet state energy transfer with the fine supramolecular cavities of proteins, the artificial triplet state photolase integrates the advantages of both the efficient reactivity of chemical photocatalysts and the precise selectivity of biocatalysts, providing an effective means to regulate the chiral selectivity of excited state reactions of organic molecules, and also fundamentally expanding the reactivity of enzyme catalysis.

The research team said that with the increasing capability of computationally assisted rational design of artificial enzymes and further development of genetic codon expansion technology, more chemical photosensitizers with unique structures and properties will be introduced into proteins to create new non-natural photoreductases, enriching the functions and applications of light-driven biocatalysis.

It is reported that Yu-Zhou Wu, Fang-Rui Zhong and Xi Chen are the co-corresponding authors of the above paper, Ning-Ning Sun and Jian-Jian Huang, Ph.D. students in the School of Chemistry and Chemical Engineering of HUST, are the co-first authors of the article, and Huazhong University of Science and Technology is the first author of the paper.

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