Selective whole-cell biosynthesis of the designer drug metabolites 15- or 16-betahydroxynorethisterone by engineered Cytochrome P450 BM3 mutants

Reinen, Jelle; Vredenburg, Galvin; Klaering, Karoline; Vermeulen, Nico; Commandeur, Jan N. M.; Honing, Maarten; Vos, J. Chris

doi:10.1016/j.molcatb.2015.08.003

Cited by 12 publications

(8 citation statements)

References 64 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In that paper, a triple and two double mutants were shown to form mainly 16β-OH-TES [8]. Since then, different BM3 variants, like M01A82W, M11A82W and M01A82WS72I, have been described as biocatalysts for steroid hydroxylation with altered regio- and stereoselectivity [6,7,11,18,19,20]. P450 BM3 variants are thus deemed good biocatalyst candidates for the oxidative hydroxylation of steroids.…”

Section: Introductionmentioning

confidence: 99%

“…The aim of previous studies was usually to identify BM3 mutants capable of hydroxylating steroids at different positions. Hence, only a few steroids, like TES [8,20], norethisterone [11,19] and progesterone [20], were used as probes to verify the hydroxylation activity of different BM3 mutants. The limited steroid substrate specificity of these BM3 mutants impedes their widespread application in the hydroxylation of steroidal precursors.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Probing Steroidal Substrate Specificity of Cytochrome P450 BM3 Variants

Liu

Wang

et al. 2016

Molecules

View full text Add to dashboard Cite

M01A82W, M11A82W and M01A82WS72I are three cytochrome P450 BM3 (CYP102A1) variants. They can catalyze the hydroxylation of testosterone (TES) and norethisterone at different positions, thereby making them promising biocatalysts for steroid hydroxylation. With the aim of obtaining more hydroxylated steroid precursors it is necessary to probe the steroidal substrate diversity of these BM3 variants. Here, three purified BM3 variants were first incubated with eight steroids, including testosterone (TES), methyltestosterone (MT), cholesterol, β-sitosterol, dehydroepiandrosterone (DHEA), diosgenin, pregnenolone and ergosterol. The results indicated that the two 3-keto-∆ 4 -steroids TES and MT can be hydroxylated at various positions by the three BM3 mutants, respectively. On the contrary, the three enzymes displayed no any activity toward the remaining six 3-hydroxy-∆ 5 -steroids. This result indicates that the BM3 mutants prefer 3-keto-∆ 4 -steroids as hydroxylation substrates. To further verify this notion, five other substrates, including two 3-hydroxy-∆ 5 -steroids and three 3-keto-∆ 4 -steroids, were carefully selected to incubate with the three BM3 variants. The results indicated the three 3-keto-∆ 4 -steroids can be metabolized to form hydroxysteroids by the three BM3 variants. On the other hand, the two 3-hydroxy-∆ 5 -steroids cannot be hydroxylated at any position by the BM3 mutants. These results further support the above conclusion, therefore demonstrating the 3-keto-∆ 4 -steroid substrate preference of BM3 mutants, and laying a foundation for microbial production of more hydroxylated steroid intermediates using BM3 variants.

show abstract

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Probing Steroidal Substrate Specificity of Cytochrome P450 BM3 Variants

Liu

Wang

et al. 2016

Molecules

View full text Add to dashboard Cite

show abstract

“…Their use for preparative biotransformation is limited primarily because of poor stability. Bacterial P450s continue to show promise for the synthesis of drug metabolites (Gillam and Hayes, 2013) with mutant enzymes from B. megaterium being particularly thoroughly investigated (Sawayama et al, 2009;Reinen et al, 2015). Both protein and process engineering approaches continue to enhance the possibilities of using these diverse catalysts at scale (Kiss et al, 2015;Venkataraman et al, 2015).…”

Section: Discussionmentioning

confidence: 99%

CYP3A Specifically Catalyzes 1 -Hydroxylation of Deoxycholic Acid: Characterization and Enzymatic Synthesis of a Potential Novel Urinary Biomarker for CYP3A Activity

Hayes

Grönberg

et al. 2016

Drug Metabolism and Disposition

View full text Add to dashboard Cite

The endogenous bile acid metabolite 1b-hydroxy-deoxycholic acid (1b-OH-DCA) excreted in human urine may be used as a sensitive CYP3A biomarker in drug development reflecting in vivo CYP3A activity. An efficient and stereospecific enzymatic synthesis of 1b-OH-DCA was developed using a Bacillus megaterium (BM3) cytochrome P450 (P450) mutant, and its structure was confirmed by nuclear magnetic resonance (NMR) spectroscopy. A [ 2 H 4 ]-labeled analog of 1b-OH-DCA was also prepared. The major hydroxylated metabolite of deoxycholic acid (DCA) in human liver microsomal incubations was identified as 1b-OH-DCA by comparison with the synthesized reference analyzed by UPLC-HRMS. Its formation was strongly inhibited by CYP3A inhibitor ketoconazole. Screening of 21 recombinant human cytochrome P450 (P450) enzymes showed that, with the exception of extrahepatic CYP46A1, the most abundant liver P450 subfamily CYP3A, including CYP3A4, 3A5, and 3A7, specifically catalyzed 1b-OH-DCA formation. This indicated that 1b-hydroxylation of DCA may be a useful marker reaction for CYP3A activity in vitro. The metabolic pathways of DCA and 1b-OH-DCA in human hepatocytes were predominantly via glycine and, to a lesser extent, via taurine and sulfate conjugation. The potential utility of 1b-hydroxylation of DCA as a urinary CYP3A biomarker was illustrated by comparing the ratio of 1b-OH-DCA:DCA in a pooled spot urine sample from six healthy control subjects to a sample from one patient treated with carbamazepine, a potent CYP3A inducer; 1b-OH-DCA:DCA was considerably higher in the patient versus controls (ratio 2.8 vs. 0.4). Our results highlight the potential of 1b-OH-DCA as a urinary biomarker in clinical CYP3A DDI studies.

show abstract

“…Reinen and co-workers screened P450BM3 libraries in vitro for the hydroxylation of the contraceptive norethisterone (NET) 40 ( Scheme 9 ). 46 Mutants “MT80” and “MT102” catalyzed regioselective transformations to 16-β-hydroxNET 41 and 15β-hydroxyNET 42 , respectively. Using whole cells of E. coli in a 1 L fed-batch system and with carbogen (80% oxygen, 20% carbon dioxide) as the oxygen supply, variant MT80 gave 47.6 mg L –1 of the 16β product 41 , whereas M102 gave 308.7 mg L –1 of the 15β product 42 , each with 99% regioselectivity.…”

Section: Cytochromes P450mentioning

confidence: 99%

Hemoprotein Catalyzed Oxygenations: P450s, UPOs, and Progress toward Scalable Reactions

Grogan

2021

JACS Au

View full text Add to dashboard Cite

The selective oxygenation of nonactivated carbon atoms is an ongoing synthetic challenge, and biocatalysts, particularly hemoprotein oxygenases, continue to be investigated for their potential, given both their sustainable chemistry credentials and also their superior selectivity. However, issues of stability, activity, and complex reaction requirements often render these biocatalytic oxygenations problematic with respect to scalable industrial processes. A continuing focus on Cytochromes P450 (P450s), which require a reduced nicotinamide cofactor and redox protein partners for electron transport, has now led to better catalysts and processes with a greater understanding of process requirements and limitations for both in vitro and whole-cell systems. However, the discovery and development of unspecific peroxygenases (UPOs) has also recently provided valuable complementary technology to P450-catalyzed reactions. UPOs need only hydrogen peroxide to effect oxygenations but are hampered by their sensitivity to peroxide and also by limited selectivity. In this Perspective, we survey recent developments in the engineering of proteins, cells, and processes for oxygenations by these two groups of hemoproteins and evaluate their potential and relative merits for scalable reactions.

show abstract

Selective whole-cell biosynthesis of the designer drug metabolites 15- or 16-betahydroxynorethisterone by engineered Cytochrome P450 BM3 mutants

Cited by 12 publications

References 64 publications

Probing Steroidal Substrate Specificity of Cytochrome P450 BM3 Variants

Probing Steroidal Substrate Specificity of Cytochrome P450 BM3 Variants

CYP3A Specifically Catalyzes 1 -Hydroxylation of Deoxycholic Acid: Characterization and Enzymatic Synthesis of a Potential Novel Urinary Biomarker for CYP3A Activity

Hemoprotein Catalyzed Oxygenations: P450s, UPOs, and Progress toward Scalable Reactions

Contact Info

Product

Resources

About