Double site saturation mutagenesis of the human cytochrome P450 2D6 results in regioselective steroid hydroxylation

Geier, Martina; Braun, Andreas; Fladischer, Patrik; Stępniak, Piotr; Rudroff, Florian; Hametner, Christian; Mihovilovic, Marko D.; Glieder, Anton

doi:10.1111/febs.12270

Cited by 21 publications

(18 citation statements)

References 37 publications

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“…The coding sequences were available from previous studies (CYP2D6/CPR 62 , CalB 30 , PDI 63 ). See also Supplementary Data 2 for the exact primers and overhangs used.…”

Section: Methodsmentioning

confidence: 99%

Engineered bidirectional promoters enable rapid multi-gene co-expression optimization

et al. 2018

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Numerous synthetic biology endeavors require well-tuned co-expression of functional components for success. Classically, monodirectional promoters (MDPs) have been used for such applications, but MDPs are limited in terms of multi-gene co-expression capabilities. Consequently, there is a pressing need for new tools with improved flexibility in terms of genetic circuit design, metabolic pathway assembly, and optimization. Here, motivated by nature’s use of bidirectional promoters (BDPs) as a solution for efficient gene co-expression, we generate a library of 168 synthetic BDPs in the yeast Komagataella phaffii (syn. Pichia pastoris), leveraging naturally occurring BDPs as a parts repository. This library of synthetic BDPs allows for rapid screening of diverse expression profiles and ratios to optimize gene co-expression, including for metabolic pathways (taxadiene, β-carotene). The modular design strategies applied for creating the BDP library could be relevant in other eukaryotic hosts, enabling a myriad of metabolic engineering and synthetic biology applications.

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“…The coding sequences were available from previous studies (CYP2D6/CPR 62 , CalB 30 , PDI 63 ). See also Supplementary Data 2 for the exact primers and overhangs used.…”

Section: Methodsmentioning

confidence: 99%

Engineered bidirectional promoters enable rapid multi-gene co-expression optimization

et al. 2018

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“…It is unlikely, though possible, that these physiological differences are related to prognosis of ER+ breast cancer. CYP2D6 has very weak affinity for testosterone[41], suggesting a possible relationship with ER+ breast cancer occurrence or prognosis, however, associations of CYP2D6 polymorphisms with occurrence of ER+ breast cancer have not been detected in very large genome-wide screens[42]. …”

Section: Discussionmentioning

confidence: 99%

CYP2D6 genotype is not associated with survival in breast cancer patients treated with tamoxifen: results from a population-based study

Hertz

Kidwell

Hilsenbeck

et al. 2017

Breast Cancer Res Treat

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Purpose: A number of studies have tested the hypothesis that breast cancer patients with low-activity CYP2D6 genotypes achieve inferior benefit from tamoxifen treatment, putatively due to lack of metabolic activation to endoxifen. Studies have provided conflicting data, and meta-analyses suggest a small but significant increase in cancer recurrence, necessitating additional studies to allow for accurate effect assessment. We conducted a retrospective pharmacogenomic analysis of a prospectively collected community-based cohort of patients with estrogen receptor-positive breast cancer to test for associations between low-activity CYP2D6 genotype and disease outcome in 500 patients treated with adjuvant tamoxifen monotherapy and 500 who did not receive any systemic adjuvant therapy. Methods: Tumor-derived DNA was genotyped for common, functionally consequential CYP2D6 polymorphisms (*2, *3, *4, *6, *10, *41 and copy number variants) and assigned a CYP2D6 activity score (AS) ranging from none (0) to full (2). Patients with poor metabolizer (PM, AS=0) phenotype were compared to patients with AS>0 and in secondary analyses AS was analyzed quantitatively. Clinical outcome of interest was recurrence free survival (RFS) and analyses using long-rank test were adjusted for relevant clinical covariates (nodal status, tumor size, etc.). Results: CYP2D6 AS was not associated with RFS in tamoxifen treated patients in univariate analyses (p>0.2). In adjusted analyses, increasing AS was associated with inferior RFS (Hazard ratio (HR)=1.43, 95% confidence interval: 1.00–2.04, p=0.05). In patients that did not receive tamoxifen treatment, increasing CYP2D6 AS, and AS>0, were associated with superior RFS (each p=0.0015). Conclusions: This population-based study does not support the hypothesis that patients with diminished CYP2D6 activity achieve inferior tamoxifen benefit. These contradictory findings suggest that the association between CYP2D6 genotype and tamoxifen treatment efficacy is null or near null, and unlikely to be useful in clinical practice.

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“…The versatility of CYP enzymes may be exploited as biocatalysts, where both bacterial [69] and mammalian [70] isoforms are under consideration. The advantage of bacterial CYPs could be the ease of expression and the higher catalytic efficiency, although the wild-type substrate specificity may be unfavorable for the majority of applications.…”

Section: Effect Of Mutationsmentioning

confidence: 99%

Structure‐Based Methods for Predicting the Sites and Products of Metabolism

Oostenbrink

2014

Methods and Principles in Medicinal Chemistry

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IntroductionCytochrome P450s (CYPs) are heme-containing enzymes that can be found in virtually all organisms. In humans, the CYP superfamily constitutes the most important enzymes in drug metabolism (see Chapters 4-8).Various computational approaches to study CYP metabolism have been reported over the years [1][2][3][4], and depending on the question that is being addressed, different methods may be more or less appropriate. For instance, if the actual reaction mechanism of a particular substrate is to be studied, a quantum mechanical description explicitly describing the electrons that are responsible for bonds being broken and formed is required [5]. On the other hand, a classification of a large number of compounds in terms of their likelihood to show an interaction with a CYP may rather be performed by application of cheminformatics machine learning tools [6]. In almost all cases, the versatility of the enzymes, the diversity of the substrates and inhibitors, and the malleability of the active sites pose additional challenges to the computational approach at hand. This chapter focuses on structure-based approaches to study the metabolism of substrates by CYP enzymes. Rather than addressing the actual enzymatic reactions, as in Chapters 6 and 11, we will focus here on the preferred orientations of substrates in the active site of CYP enzymes as a crucial first step in the prediction of metabolism. Using mostly examples from our own work, we will highlight some of the possibilities and challenges in structure-based predictions of sites of metabolism (SoMs). 6 Å RuleThe basic concept behind structure-based prediction of metabolism by CYP enzymes is extremely simple. The crucial cofactor of the enzymes is a heme 243 Drug Metabolism Prediction, First Edition. Edited by Johannes Kirchmair.

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Double site saturation mutagenesis of the human cytochrome P450 2D6 results in regioselective steroid hydroxylation

Cited by 21 publications

References 37 publications

Engineered bidirectional promoters enable rapid multi-gene co-expression optimization

Engineered bidirectional promoters enable rapid multi-gene co-expression optimization

CYP2D6 genotype is not associated with survival in breast cancer patients treated with tamoxifen: results from a population-based study

Structure‐Based Methods for Predicting the Sites and Products of Metabolism

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