Characterization of a thermophilic cytochrome P450 of the CYP203A subfamily from Binh Chau hot spring in Vietnam

Nguyen, Kim-Thoa; Nguyen, Ngọc-Lan; Milhim, Mohammed; Nguyen, Van-Tung; Lai, Thi-Hong-Nhung; Nguyen, Huy-Hoang; Le, Thi-Thanh-Xuan; Phan, Thi-Tuyet-Minh; Bernhardt, Rita

doi:10.1002/2211-5463.13033

Cited by 5 publications

(3 citation statements)

References 46 publications

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“…It is noteworthy, that a second transition was evident in the melting curve of the purified bovine enzyme, which might derive from the previously described contamination with a small fraction of the molecular chaperone GroEL. However, the T m value of the vertebrate ancestor CYP11A_N1 was strikingly increased by 25 °C to 74.2 ± 0.4 °C and even exceeded the reported melting temperatures of some naturally thermostable P450s including CYP154H1 (67 °C) from Thermobifida fusca [48], CYP231A2 (65 °C) from Picrophilus torridus [49] or CYP450‐T2 (56.8 °C), a not‐yet‐classified P450 derived from metagenomic data of the Binh Chau hot spring [50]. In contrast to these P450s, CYP11A_N1 was not likely to have existed in a hot environment where thermal robustness was required, given that it is expected to have evolved around the time vertebrates emerged on earth and where temperatures were similar to those presently [9].…”

Section: Resultsmentioning

confidence: 95%

Resurrection and characterization of ancestral CYP11A1 enzymes

et al. 2021

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Mitochondrial cytochromes P450 presumably originated from a common microsomal P450 ancestor. However, it is still unknown how ancient mitochondrial P450s were able to retain their oxygenase function following relocation to the mitochondrial matrix and later emerged as enzymes specialized for steroid hormone biosynthesis in vertebrates. Here, we used the approach of ancestral sequence reconstruction (ASR) to resurrect ancient CYP11A1 enzymes and characterize their unique biochemical properties. Two ancestral CYP11A1 variants, CYP11A_Mammal_N101 and CYP11A_N1, as well as an extant bovine form were recombinantly expressed and purified to homogeneity. All enzymes showed characteristic P450 spectral properties and were able to convert cholesterol as well as other sterol substrates to pregnenolone, yet with different specificities. The vertebrate CYP11A_N1 ancestor preferred the cholesterol precursor, desmosterol, as substrate suggesting a convergent evolution of early cholesterol metabolism and CYP11A1 enzymes. Both ancestors were able to withstand increased levels of hydrogen peroxide but only the ancestor CYP11A_N1 showed increased thermostability (~25°C increase in T 50 ) compared with the extant CYP11A1. The extraordinary robustness of ancient mitochondrial P450s, as demonstrated for CYP11A_N1, may have allowed them to stay active when presented with poorly compatible electron transfer proteins and resulting harmful ROS in the new environment of the mitochondrial matrix. To the best of our knowledge, this work represents the first study that describes the resurrection of ancient mitochondrial P450 enzymes. The results will help to understand and gain fundamental functional insights into the evolutionary origins of steroid hormone biosynthesis in animals.

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Section: Resultsmentioning

confidence: 95%

Resurrection and characterization of ancestral CYP11A1 enzymes

et al. 2021

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“…Thermophilic enzymes, such as CYP116B29, CYP116B46, CYP116B64, CYP116B65, CYP119, CYP119A1, CYP119A2, CYP154H1, CYP174A1, CYP175A1, CYP174A1, and CYP231A2 can be used at higher temperature than normal for enzymes. [151][152][153] The thermal stability of these enzymes makes them attractive for industrial use. For example, a CYP119/sol-gel film remained stable up to 60 1C, but the reduction current decreased to B45% as compared to that at 30 1C.…”

Section: Challenges In Enzyme Electrocatalysis and Their Solutionsmentioning

confidence: 99%

Electrochemical transformations catalyzed by cytochrome P450s and peroxidases

Kumar

Rusling

2023

Chem. Soc. Rev.

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“…[29] They obtained 12500 nmol per liter, which is a similar amount to this study (12.5 to 16.7 μmol per liter). In another study, E. coli C43 (DE3) was the most suitable host for the expression of CYP203A1 from Rhodopseudomonas palustris which was not self-sufficient [30] Parikh and Guengerich found that 28 °C and 48 hours are optimal conditions for artificially generated self-sufficient human P450 1A2 fused to rat P450 reductase. [31]…”

Section: Enhanced Expression Of the P450 Azc1mentioning

confidence: 99%

Expression and Characterization of a New Self‐Sufficient P450 Monooxygenase (P450 AZC1) from Azorhizobium caulinodans

Yıldırım,

Ozic,

Ensari

2023

ChemBioChem

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Oxyfunctionalization of non-activated carbon bonds by P450 monooxygenases has drawn great industrial attraction. Selfsufficient P450s containing catalytic heme and reductase domains in a single polypeptide chain offer many advantages since they do not require external electron transfer partners.Here, we report the first P450 enzyme identified and expressed from Azorhizobium caulinodans. Firstly, expression conditions of P450 AZC1 were optimized for enhanced expression in E.coli. The highest P450 content was obtained in E.coli Rosetta DE3 plysS when it was incubated in TB media supplemented with 0.75 mM IPTG, 0.5 mM ALA, and 0.75 mM FeCl 3 at 25 °C for 24 hours. Subsequently, the purified enzyme showed a broad substrate spectrum including fatty acids, linear and cyclic alkanes, aromatics, and pharmaceuticals. Finally, P450 AZC1 showed optimal activity at pH 6.0 and 40 °C and a broad pH and temperature profile, making it a promising candidate for industrial applications.

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Characterization of a thermophilic cytochrome P450 of the CYP203A subfamily from Binh Chau hot spring in Vietnam

Cited by 5 publications

References 46 publications

Resurrection and characterization of ancestral CYP11A1 enzymes

Resurrection and characterization of ancestral CYP11A1 enzymes

Electrochemical transformations catalyzed by cytochrome P450s and peroxidases

Expression and Characterization of a New Self‐Sufficient P450 Monooxygenase (P450 AZC1) from Azorhizobium caulinodans

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