Important progress has been made in the design of genetically encoded phosphine ligands and artificial metalloproteins (enzymes).

On November 23, 2022, the journal JACS published a research paper entitled “Genetically Encoded Phosphine Ligand for Metalloprotein Designn” by Wang Jiangyun’s research group and Chen Yongxiang’s research group of Tsinghua University, which introduced unnatural phosphine ligands into living systems through the genetic codedon extension (GCE) strategy for the first time, and then developed the construction strategy of artificial metalloproteins (enzymes) containing unnatural phosphine ligands.

In nature and organisms, as an important element that constitutes life, phosphorus mostly exists in living systems in the form of +5 valence compounds and plays vital physiological functions. +3 valence phosphorus has richer functional properties due to its special electronic structure, but due to the limitations of the earth’s biosphere in an oxygen-rich environment, organisms have hardly evolved operating laws that can use low-oxidized phosphorus. Chemists can develop +3 valence phosphorus into an important catalyst ligand other than N, O and S elements, that is, phosphine ligands, and chemically evolve transition metal catalysts with special catalytic activity, develop many reaction types that do not exist in nature, extend these achievements and wisdom to biological evolution systems, and then design and construct new artificial metal proteins, which is a promising research direction in synthetic biology.

How to introduce unnatural phosphine ligands into living systems

This work overcomes three major challenges in genetically coding phosphine ligands: 1) phosphine ligands have strong oxygen sensitivity and are incompatible with the directed evolution of aminoacyl-tRNA synthetase under aerobic conditions, intracellular protein biosynthesis processes and physiological conditions; 2) The metal coordination and conjugation process in metal-organic chemistry is mostly carried out in the glove box with an inert atmosphere, and it is difficult to prepare artificial metalloproteins (enzymes) containing phosphine ligands under ordinary biochemical experimental conditions; 3) The phosphine ligand structure generally has a large volume and poor water solubility. For the first time, low-valent, unnatural phosphine ligands were introduced into living systems through GCE strategies and inserted into arbitrary sites on the protein skeleton.

The researchers first designed and synthesized a borane-protected phosphine-containing ligand unnatural amino acid P3BF, which is relatively stable in aqueous phase and air conditions. After screening and directed evolution, an aminoacyl-tRNA synthase mutant P3BFRS that can specifically recognize P3BF was obtained. With the help of genetic codon extension technology, P3BF is inserted into any specific site of the protein within E. coli; For the first time, the crystal structure of proteins with unnatural phosphine-boron (P-B) bonds with a resolution of 1.95 Å was resolved.

In addition, the researchers developed a new strategy that can directly convert phosphoboron compound P3BF into phospho-metal complex in one simple step under aerobic conditions of aqueous phase and air, which is mild, efficient, green, compatible with proteins, easy to operate, and does not require anaerobic treatment, providing a feasible scheme for chemical conversion on proteins; According to the experimental phenomenon, the mechanism of the direct transformation reaction of the one-step method is preliminarily explored, and the possible reaction paths are given. On this basis, this paper inserts phosphoboron unnatural amino acid P3BF into an LmrR protein with a special cavity structure, and uses the one-step direct conversion strategy to construct an artificial metalloprotein containing phosphine-cyclopalladium metal complex under the conditions of aqueous phase and air, and develops a new strategy for phosphine-metalloprotein construction, which creates favorable conditions for the next step to achieve artificial metalloenzymes and even whole-cell catalysis.

In this paper, the introduction of non-natural phosphine ligands and the construction strategy of artificial metalloproteins

Dr. Hu Cheng, a postdoctoral fellow in the station, and Huazhen Duan, a doctoral student at Tsinghua University, are the co-first authors of the paper, and Professor Wang Jiangyun, Professor Liu Xiaohong and Associate Professor Chen Yongxiang are the co-corresponding authors of the paper. This work has been funded and supported by the Key Research and Development Program of the Ministry of Science and Technology, the National Natural Science Foundation of China and other funds. (Source: Institute of Biophysics, Chinese Academy of Sciences)

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