Characterization of two steroid hydroxylases from different <i>Streptomyces</i> spp. and their ligand‐bound and ‐unbound crystal structures

Dangi, Bikash; Lee, Chang Woo; Kim, Ki-Hwa; Park, Sunha; Yu, Eun-Ji; Jeong, Chang-Sung; Park, Hyun; Lee, Jun Hyuck; Oh, Tae‐Jin

doi:10.1111/febs.14729

Cited by 13 publications

(13 citation statements)

References 61 publications

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“…As for other biocatalysts, a bacterial P450 CYP102A1 (P450-BM3) mutant F87V was reported to have 2␤and 15␤-hydroxylase activities toward progesterone and testosterone (43). Recently, two other bacterial hydroxylases, CYP154C4-1 and CYP154C4-2, have been reported to convert testosterone to 2␣-and 2␤-hydroxylated products (15). Interestingly, they showed hydroxylation activities mainly at the 16␣ position when progesterone was used as the substrate.…”

Section: Discussionmentioning

confidence: 99%

“…The CYP109 family member CYP109E1 has been characterized as a 16␤-hydroxylase of 3-ketosteroids (12). CYP154C family P450s show 16␣-hydroxylation activities toward various steroidal substrates (13)(14)(15). CYP260A1 of the CYP260A family of P450s is able to convert various C 19 steroids at the 1␣ position, while CYP260B1 has been identified as a 6␤-and 9␣-hydroxylase of C 21 steroids (16,17).…”

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confidence: 99%

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Distinct Regioselectivity of Fungal P450 Enzymes for Steroidal Hydroxylation

Lü

Feng

Chen

et al. 2019

Appl Environ Microbiol

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In this study, we identified two P450 enzymes (CYP5150AP3 and CYP5150AN1) from Thanatephorus cucumeris NBRC 6298 by combination of transcriptome sequencing and heterologous expression in Pichia pastoris. The biotransformation of 11-deoxycortisol and testosterone by Pichia pastoris whole cells coexpressing the cyp5150ap3 and por genes demonstrated that the CYP5150AP3 enzyme possessed steroidal 7β-hydroxylase activities toward these substrates, and the regioselectivity was dependent on the structures of steroidal compounds. CYP5150AN1 catalyzed the 2β-hydroxylation of 11-deoxycortisol. It is interesting that they display different regioselectivity of hydroxylation from that of their isoenzyme, CYP5150AP2, which possesses 19- and 11β-hydroxylase activities. IMPORTANCE The steroidal hydroxylases CYP5150AP3 and CYP5150AN1 together with the previously characterized CYP5150AP2 belong to the CYP5150A family of P450 enzymes with high amino acid sequence identity, but they showed completely different regioselectivities toward 11-deoxycortisol, suggesting the regioselectivity diversity of steroidal hydroxylases of CYP5150 family. They are also distinct from the known bacterial and fungal steroidal hydroxylases in substrate specificity and regioselectivity. Biocatalytic hydroxylation is one of the important transformations for the functionalization of steroid nucleus rings but remains a very challenging task in organic synthesis. These hydroxylases are useful additions to the toolbox of hydroxylase enzymes for the functionalization of steroids at various positions.

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

Distinct Regioselectivity of Fungal P450 Enzymes for Steroidal Hydroxylation

Lü

Feng

Chen

et al. 2019

Appl Environ Microbiol

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“…ATCC 11861 (StCYP154C4-2) can produce isomers of 2-hydroxy-TST. [81] Although CYP154C4-1 and CYP154C4-2 share 93% sequence identity and only 29 different residues, there are some differences existing between them. In the reaction of 3, CYP154C4-2 showed complete conversion to 2-OH-TST (mixture of 2α and 2β), and CYP154C4-1 produced a few byproducts, while CYP154C4-1 was more tightly bound to 3 than CYP154C4-2.…”

Section: Cyp154 Familymentioning

confidence: 99%

“…In the reaction of 3, CYP154C4-2 showed complete conversion to 2-OH-TST (mixture of 2α and 2β), and CYP154C4-1 produced a few byproducts, while CYP154C4-1 was more tightly bound to 3 than CYP154C4-2. [81] To date, several crystal structures of the CYP154C subfamily have been solved, such as CYP154C5steroid (26, 1, 2, 3) structures, [78] substrate-free CYP154C4 and in complex with 3, [81] substrate-free CYP154C2, [76] and the crystal structure of CYP154C1 closely related to CYP154C5 [82] that can be used as general model for the substrate-free CYP154C5 structure. All of them contain a larger hydrophobic region corresponding to the backbone of the steroids and show a relatively narrow and closed substrate pocket, likely contributing to its strict substrate selectivity.…”

Section: Cyp154 Familymentioning

confidence: 99%

Cytochrome P450 Monooxygenases Catalyse Steroid Nucleus Hydroxylation with Regio‐ and Stereo‐Selectivity

et al. 2022

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Steroids are the second largest class of drugs with a wide range of pharmacological properties. Hydroxylation of steroids seriously affects their biological activities and other properties. However, steroids are mostly sp 3 hybridized carbons with numerous CÀ H bonds far from the functional group that can activate them, and achieving regio-and stereo-selective hydroxylation on steroids is a highly challenging task that is almost impossible to achieve using modern organic synthesis techniques. Interestingly, cytochrome P450 monooxygenases possess the ability to catalyse regio-and stereo-selective oxidations of nonactivated CÀ H bonds in complex organic molecules under mild conditions. This review summarizes the P450s identified and engineered in recent years that can catalyse steroid nucleus hydroxylation stereo-and regio-selectively.1.

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“…Bacterial monooxygenases compared to those in eukaryotes are advantageous for biotechnological applications because of their solubility and high level of expression [21]. A few CYPs are involved in the hydroxylation of steroids, like the CYP106A family (CYP106A1 and CYP106A2), the CYP109 family (CYP109B1 and CYP109E1), the CYP154C family (CYP154C2, CYP154C3, CYP154C4, CYP154C5, and CYP154C8), and CYP260A1 [17,[22][23][24][25][26][27].…”

Section: Introductionmentioning

confidence: 99%

Enzymatic Characterization and Comparison of Two Steroid Hydroxylases CYP154C3-1 and CYP154C3-2 fromStreptomycesSpecies

Subedi

Kim

Hong

et al. 2021

J. Microbiol. Biotechnol.

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Bacterial cytochrome P450 (CYP) enzymes are responsible for the hydroxylation of diverse endogenous substances with a heme molecule used as a cofactor. This study characterized two CYP154C3 proteins from Streptomyces sp. W2061 (CYP154C3-1) and Streptomyces sp. KCCM40643 (CYP154C3-2). The enzymatic activity assays of both CYPs conducted using heterologous redox partners' putidaredoxin and putidaredoxin reductase showed substrate flexibility with different steroids and exhibited interesting product formation patterns. The enzymatic characterization revealed good activity over a pH range of 7.0 to 7.8 and the optimal temperature range for activity was 30 to 37°C. The major product was the C16-hydroxylated product and the kinetic profiles and patterns of the generated hydroxylated products differed between the two enzymes. Both enzymes showed a higher affinity toward progesterone, with CYP154C3-1 demonstrating slightly higher activity than CYP154C3-2 for most of the substrates. Oxidizing agents (diacetoxyiodo) benzene (PIDA) and hydrogen peroxide (H 2 O 2 ) were also utilized to actively support the redox reactions, with optimum conversion achieved at concentrations of 3 mM and 65 mM, respectively. The oxidizing agents affected the product distribution, influencing the type and selectivity of the CYP-catalyzed reaction. Additionally, CYP154C3s also catalyzed the C-C bond cleavage of steroids. Therefore, CYP154C3s may be a good candidate for the production of modified steroids for various biological uses.

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Characterization of two steroid hydroxylases from different Streptomyces spp. and their ligand‐bound and ‐unbound crystal structures

Cited by 13 publications

References 61 publications

Distinct Regioselectivity of Fungal P450 Enzymes for Steroidal Hydroxylation

Distinct Regioselectivity of Fungal P450 Enzymes for Steroidal Hydroxylation

Cytochrome P450 Monooxygenases Catalyse Steroid Nucleus Hydroxylation with Regio‐ and Stereo‐Selectivity

Enzymatic Characterization and Comparison of Two Steroid Hydroxylases CYP154C3-1 and CYP154C3-2 fromStreptomycesSpecies

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