A P450 fusion library of heme domains from  Rhodococcus jostii  RHA1 and its evaluation for the biotransformation of drug molecules

Kulig, Justyna; Spandolf, Claudia; Hyde, Ralph J.; Ruzzini, Antonio C.; Eltis, Lindsay D.; Grönberg, Gunnar; Hayes, Martin A.; Grogan, Gideon

doi:10.1016/j.bmc.2015.07.025

Cited by 21 publications

(24 citation statements)

References 28 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…It has been shown recently that several members of a P450 fusion library, constructed by P450 enzymes and their autologous redox system RhfRED from R. jostii RHA1, are able to convert five out of 48 selected drugs (Kulig et al, 2015). Compared with our system consisting of the wild-type CYP267B1 and its autologous redox partners Fdx8 and FdR_B, we observed a larger substrate range (14 out of 22 drugs) and significantly higher activity toward the conversion of drugs.…”

Section: Discussionmentioning

confidence: 63%

“…2; Supplemental Table 2). These bacterial P450 enzymes are considered to be active drug metabolizers for several drugs including amitriptyline, chlorpromazine (2), and diclofenac (5) (Prior et al, 2010;Kulig et al, 2015). In addition, we have previously shown that CYP264A1 was able to convert tricyclic drug molecules (Litzenburger et al, 2015), and CYP265A1 and CYP266A1 were able to hydroxylate the antitumor drug epothilone D .…”

Section: Bioinformatics Studies and Comparison Of Cyp267a1 With Cyp267b1mentioning

confidence: 99%

See 1 more Smart Citation

CYP267A1 and CYP267B1 from Sorangium cellulosum So ce56 are Highly Versatile Drug Metabolizers

Kern

Khatri

Litzenburger

et al. 2016

Drug Metabolism and Disposition

View full text Add to dashboard Cite

The guidelines of the Food and Drug Administration and International Conference on Harmonization have highlighted the importance of drug metabolites in clinical trials. As a result, an authentic source for their production is of great interest, both for their potential application as analytical standards and for required toxicological testing. Since we have previously shown promising biotechnological potential of cytochromes P450 from the soil bacterium Sorangium cellulosum So ce56, herein we investigated the CYP267 family and its application for the conversion of commercially available drugs including nonsteroidal anti-inflammatory, antitumor, and antihypotensive drugs. The CYP267 family, especially CYP267B1, revealed the interesting ability to convert a broad range of substrates. We established substrate-dependent extraction protocols and also optimized the reaction conditions for the in vitro experiments and Escherichia coli-based whole-cell bioconversions. We were able to detect activity of CYP267A1 toward seven out of 22 drugs and the ability of CYP267B1 to convert 14 out of 22 drugs. Moderate to high conversions (up to 85% yield) were observed in our established whole-cell system using CYP267B1 and expressing the autologous redox partners, ferredoxin 8 and ferredoxin-NADP + reductase B. With our existing setup, we present a system capable of producing reasonable quantities of the human drug metabolites 49-hydroxydiclofenac, 2-hydroxyibuprofen, and omeprazole sulfone. Due to the great potential of converting a broad range of substrates, wild-type CYP267B1 offers a wide scope for the screening of further substrates, which will draw further attention to future biotechnological usage of CYP267B1 from S. cellulosum So ce56.

show abstract

Section: Discussionmentioning

confidence: 63%

Section: Bioinformatics Studies and Comparison Of Cyp267a1 With Cyp267b1mentioning

confidence: 99%

CYP267A1 and CYP267B1 from Sorangium cellulosum So ce56 are Highly Versatile Drug Metabolizers

Kern

Khatri

Litzenburger

et al. 2016

Drug Metabolism and Disposition

View full text Add to dashboard Cite

show abstract

“…A few CYPs are known to display activity in the presence of (diacetoxyiodo)benzene. Mammalian and bacterial CYPs such as CYP5A1, CYP17A1, CYP121, CYP101A1 (P450 cam ), and CYP106A2 are known to show catalytic activity in the presence of iodosobenzene as a single‐oxygen donor . The mechanism of oxygenation supported by (diacetoxyiodo)benzene might be similar to that supported by iodosobenzene, probably mediated by two‐electron transfer of a single oxygen atom from oxidant to ferric CYP to generate compound I, which is involved in substrate monooxygenation .…”

Section: Discussionmentioning

confidence: 99%

“…However, few CYPs are known to display such characteristics in the presence of specific chemical compounds. The characterization or synthetic application of CYPs has often been mediated through one or more surrogate redox partners either in an isolated form or after artificial fusion with the CYP complex, due to the difficulty in obtaining native redox partners . It is generally believed that the choice of surrogate partners or their mode of action does not affect the type and selectivity of the reactions catalyzed by CYPs .…”

Section: Introductionmentioning

confidence: 99%

Effects of Alternative Redox Partners and Oxidizing Agents on CYP154C8 Catalytic Activity and Product Distribution

Dangi

Park

2018

ChemBioChem

View full text Add to dashboard Cite

CYP154C8 catalyzes the hydroxylation of diverse steroids, as has previously been demonstrated, by using an NADH-dependent system including putidaredoxin and putidaredoxin reductase as redox partner proteins carrying electrons from NADH. In other reactions, CYP154C8 reconstituted with spinach ferredoxin and NADPH-dependent ferredoxin reductase displayed catalytic activity different from that of the NADH-dependent system. The NADPH-dependent system showed multistep oxidation of progesterone and other substrates including androstenedione, testosterone, and nandrolone. (Diacetoxyiodo)benzene was employed to generate compound I (FeO ), actively supporting the redox reactions catalyzed by CYP154C8. In addition to 16α-hydroxylation, progesterone and 11-oxoprogesterone also underwent hydroxylation at the 6β-position in reactions supported by (diacetoxyiodo)benzene. CYP154C8 was active in the presence of high concentrations (>10 mm) of H O , with optimum conversion surprisingly being achieved at ≈75 mm H O . More importantly, H O tolerance by CYP154C8 was evident in the very low heme oxidation rate constant (K) even at high concentrations of H O . Our results demonstrate that alternative redox partners and oxidizing agents influence the catalytic efficiency and product distribution of a cytochrome P450 enzyme. More importantly, these choices affected the type and selectivity of reaction catalyzed by the P450 enzyme.

show abstract

“…This requirement hinders practical applications of the P450s. To overcome this inconvenience, artificial “self‐sufficient” P450s, in which redox proteins are arranged near a P450 moiety and supply electrons to it intramolecularly, have been developed, and chimeric constructs containing non‐natural redox proteins have increased the versatility of bacterial P450s …”

Section: Introductionmentioning

confidence: 99%

Artificial Self‐Sufficient Cytochrome P450 Containing Multiple Auxiliary Proteins Demonstrates Improved Monooxygenase Activity

Haga

Hirakawa

Nagamune

2018

Biotechnology Journal

View full text Add to dashboard Cite

Most bacterial cytochrome P450 monooxygenases (P450s) do not work alone because their active species is generated by two electrons supplied through two separate auxiliary proteins. Artificial "self-sufficient" P450s, in which one molecule each of the two auxiliary proteins is arranged close to the P450s, have been developed but have not achieved the maximum catalytic turnover numbers of the P450s. In this study, the Pseudomonas putida P450 (P450cam) is assembled with multiple molecules of its auxiliary proteins, putidaredoxin (PdX) and putidaredoxin reductase (PdR), by fusion to a heterotrimeric protein. In the assembled P450cam containing one PdX and one PdR, kinetic analysis reveales that the catalytic cycle of P450cam is suspended twice awaiting the reduction of PdX by PdR. An increase in the number of PdR molecules stimulated the PdX reduction process. Assembly with two PdXs allows one PdX to be reduced during the binding of the other PdX to P450cam for the first electron transfer, eliminating one waiting step. Finally, P450cam assembled with two PdXs and three PdRs showes 92% of the maximum activity of free P450cam. Therefore, assembly with multiple molecules of auxiliary proteins will facilitate in vitro biotechnological applications of the P450s.

show abstract

A P450 fusion library of heme domains from Rhodococcus jostii RHA1 and its evaluation for the biotransformation of drug molecules

Cited by 21 publications

References 28 publications

CYP267A1 and CYP267B1 from Sorangium cellulosum So ce56 are Highly Versatile Drug Metabolizers

CYP267A1 and CYP267B1 from Sorangium cellulosum So ce56 are Highly Versatile Drug Metabolizers

Effects of Alternative Redox Partners and Oxidizing Agents on CYP154C8 Catalytic Activity and Product Distribution

Artificial Self‐Sufficient Cytochrome P450 Containing Multiple Auxiliary Proteins Demonstrates Improved Monooxygenase Activity

Contact Info

Product

Resources

About