The full-length crystal structure of the 10-power biocatalyst has been cracked

On May 29th Nature Communications published online the first detailed three-dimensional structure of the cytochrome P450 enzyme, answering a scientific conundrum that has remained unresolved for decades.
Chen Chunqi, Ma Lixin and Guo Ruiting, professors of the National Key Laboratory for Biocatalysis and Enzyme Engineering at Hubei University's School of Life Sciences and provincial departments, completed the work. They analyzed the crystal structure of a P450 enzyme, CYP116B46, and clarified how electrons are transmitted within molecules. This provides important guidance for understanding the structure and complete structure of P450, and is of great value to the application of P450 enzymes in medicine and industry.cytochrome P450 is an enzyme protein first found in liver cells in mice in 1955 because its reduced-state absorption spectral wavelength in combination with carbon monoxide is about 450 nm.
the protein consists of a hemoglobin (heme) domain and a reductase domain containing a single nucleotide (FMN) of the keratin; "Cytochrome P450 is a large family of enzymes, referred to simply as CYP, involved in the synthesis of many natural products and toxic, drug metabolic reactions." In plants, the P450 family is involved in the synthesis of special compounds, such as artemisinin and yew alcohol, and in humans, the P450 family is mainly involved in detoxification processes, such as dissolving aflatoxin, which can cause cancer, Chen Chunqi, the paper's author, told China Science Daily.
this is due to the high catalytic multi-energy of P450, involving more than 20 types of reactions, including hydroxylation reactions, cyclooxidation reactions and cyclic reactions. Its substrate spectrum is very broad, can identify aromatic family, poly ketones, terpenes, peptides, sugars and other types of substrates, with "all-in-one biocatalyst" called.
it also has a high degree of regional selectivity and stereo selectivity, can specifically identify structural bits and directions. In addition, the base binding area of the P450 enzyme is highly malleable and can be modified by enzyme engineering to alter the substrate spectrum, with great potential for applications in important chemicals and drug manufacturing.
, drug activity and metabolism are primarily the responsibility of the CYP family of enzymes, which account for about 75 percent of such metabolism in organisms.
P450 system is widely found in all living organisms and even viruses, and more than 18,000 P450 systems have been found. More than 57 P450 systems have been found in the human body, mostly membrane proteins, mainly distributed in the membrane or endosurgen network of the granules. However, no trace of this enzyme protein has been found in E. coli.
"the vast majority of P450 enzymes require redoxases to provide electrons to instile hemohemolyin in the substrate binding area, which in turn transforms the substrate." Chen Chunqi said that building a P450 system must look for the same source or matching redoxase.
of the nine families in the P450 system, only two have their own redoxase, known as the self-sufficient P450 enzyme, which comes mainly from bacteria, on the same peptide chain. Other P450 systems require a matching redoxase from the outside world, which occurs mainly in animals.
"s self-sufficient P450 enzyme is an attractive biocatalyst for biotechnology applications. Therefore, how to transfer electrons inside the self-sufficient P450 enzyme is a very interesting and important subject. Guo Ruiting, author of the paper, told China Science daily that scientists had previously studied areas such as hemolyte and FMN in the P450 system, but had never understood the intermediate connection and lacked a full-length fine structure.
" in the process of protein purification and culture crystals, P450 enzyme is very easy to break or degrade from the middle, so it is difficult to obtain a complete full-length three-dimensional structure. Chen Chunqi said.two types of self-sufficient P450 enzymes are currently known. The first category is represented by CYP102A1 derived from Bacillus spores, consisting of the hemohemolytic domain of the nitrogen end and the P450 reductase (CPR) of the carbon end, and the direction of electron transmission is from the carbon end to the nitrogen end. Although the full-length fine structure of this type of P450 enzyme is still missing, there are some rough theories about the electron transfer of this type of P450 enzyme.the second category is CYP116, and the CYP116B46 involved in this study falls into this category. This type of P450 consists of the heme domain of nitrogen and the phthalates of carbon end phthalates reductase (PDR), which contains the reductase domain of FMN-dependent and ferrinogenic structure.electrons are passed from carbon to hemohemolytin at the nitrogen end in a polypeptide chain of self-sufficient P450 enzymes? Because of the chronic lack of information on the structure of the protein for more than 30 years, it was not clear before the study.
, a professor of chemical biology at the University of Manchester in the United Kingdom. "Getting full-length enzymes is a great achievement," said Sabine L. Flitsch. "Many groups, including her own, have tried to parse these structures because of their significant application potential as biocatalysts.
" can be said that the crystal system has gone beyond science, almost become an art. Chen Chunqi said the difficulty of obtaining a complete large protein crystal structure is predictable.
the relative stability of the CYP116B46 used in this study, coupled with President Guo Rui's extensive experience in crystal system preparation and structural analysis, they were able to use X-ray crystallology to analyze its complete crystal structure.
structural analysis shows that in this enzyme, FMN is close enough to the iron sulfur cluster to achieve direct electron transfer, but hemohemolyte is too far away from the iron sulfur cluster to allow direct electron transfer, but rely on five "second passer" amino acids to achieve transmission.
" we analyzed the crystal structure from the full length of CYP116B46, and established a clear enzyme structure model. Guo Ruiting said that this continuous polypeptide is folded into three domains, connected by two connected peptides, from the nitrogen end to the carbon end in sequence: hemoglobin binding domain, reductase domain and ferrooxygen structure domain.
Guo Ruiting, the structure analysis found that the arrangement of the three domains of CYP116B46 and the direction of electron transmission is very consistent. The substrate binding region of hemolybin binding domain is outward-facing, which is conducive to the substrate entering the binding, which shows that the arrangement of the structure is quite reasonable. Among them, the straight-line distance between the reductase domain and the ferrobrotein domain is 7.9 ,000, which is conducive to the direct transmission of electrons.
"scientists generally believe that distances less than 8 s are directly transmitted by electrons to the past. Guo Ruiting said.
other hand, the straight-line distance between ferrepin and hemoglobin is 25.3 , which exceeds the effective direct electron transmission distance currently recognized." between the two redox centers, it is less likely that other cofactors will help with electron transport. Guo Ruiting explains that because the structure shows a narrow channel between the two, there is a lack of other cofactors combined characteristics. On the other hand, considering that amino acids are also an electronic carrier, it is also possible that amino acids in between may play an important role in the process of electronic transmission.
now have the opportunity to design reductase domains and electron transfer pathways," he said. Chen Chunqi said.
to analyze the effects of side-chain groups of these amino acids on the activity of the CYP116B46 enzyme, they mutated these bits into alanine and measured the amount of product production to assess the role of each amino acid.
Guo Ruiting, the results of the enzyme test showed a significant decrease in the activity of alanine mutants in R388, R718, E723, S726 and E729. These bits are re-labeled to the structure, and the approximate direction of electron transmission can be seen.
red caused by the lack of hemolybin in the R378A mutant protein, and no absorption peak band shift appeared after carbon monoxide treatment. Coupled with the proximity of R378 to hemolyin, it is speculated that R378 may be associated with binding hemolybin. Therefore, the effect of the mutation of R378A is more complex, and further experiments are needed to determine the effect of this bit on CYP116B46 electron transmission.know the role of amino acids in the electron delivery process, we can consider looking for alternative effective amino acids to improve the efficiency of electron transfer. Guo Ruiting explains that it's like changing hands to a longer "second passer" that allows electrons to pass faster.
design will improve the efficiency of catalytic reactions. The analysis of P450's full-length structure is a very important milestone for understanding the catalytic process of self-sufficient P450 enzyme, and it is of great guiding significance for P450 enzyme modification and application.
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