The Role of Protein Plasticity in Computational Rationalization Studies on Regioselectivity in Testosterone Hydroxylation by Cytochrome P450 BM3 Mutants

Beer, Stephanie B.A. de; Bergen, Laura A. H. van; Keijzer, Karlijn; Rea, Vanina; Venkataraman, Harini; Guerra, Célia Fonseca; Bickelhaupt, F. Matthias; Vermeulen, Nico; Commandeur, Jan N. M.; Geerke, Daan P.

doi:10.2174/138920012798918471

Cited by 11 publications

(4 citation statements)

References 15 publications

(32 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The application of the high-scoring poses has proved to be effective in the SOM prediction of specific CYPs substrates, such as the metabolism of tramadol in CYP2D6 and the metabolism of testosterone in P450 BM3. 57,58 However, our results revealed that their positive results were in some special cases. There were only ∼30% of tested CYP3A4 substrates that could position the SOM at the closest place to Fe within the top three poses (Table 5).…”

Section: Discussionmentioning

confidence: 99%

Prediction of Sites of Metabolism of CYP3A4 Substrates Utilizing Docking-Derived Geometric Features

Feng

Gong

Zhu

et al. 2023

J. Chem. Inf. Model.

View full text Add to dashboard Cite

Cytochrome P450 3A4 (CYP3A4) is one of the major drug-metabolizing enzymes in the human body and is responsible for the metabolism of ∼50% of clinically used drugs. Therefore, the identification of the compound’s sites of metabolism (SOMs) mediated by CYP3A4 is of utmost importance in the early stage of drug discovery and development. Herein, docking-based approaches incorporating geometric features were used for SOMs prediction of CYP3A4 substrates. The cross-docking poses of a relatively large data set containing 474 substrates were analyzed in depth, and a widely observed geometric pattern called the close proximity of SOMs was derived from the poses. On the basis of the close proximity, several structure-based models have been constructed, which demonstrated better performance than those structure-based models using the criterion of Fe-SOM distance. For further improving the prediction performance, the structure-based models were also combined with the well-known ligand-based model SMARTCyp. One combined model exhibited good performance on the SOMs prediction of an external substrate set containing kinase inhibitors, PROTACs, approved drugs, and some lead compounds.

show abstract

Section: Discussionmentioning

confidence: 99%

Prediction of Sites of Metabolism of CYP3A4 Substrates Utilizing Docking-Derived Geometric Features

Feng

Gong

Zhu

et al. 2023

J. Chem. Inf. Model.

View full text Add to dashboard Cite

show abstract

“…The concept of ensemble docking has since then been applied more often to CYP enzymes, in particular for the bacterial CYP102A1 (CYP BM3) [49] and for the human isoforms 2D6 [50] and 3A4 [51]. In the latter study, 16 substrates were docked into 125 protein structures as obtained from MD simulations.…”

Section: Protein Flexibilitymentioning

confidence: 99%

“…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. Various drug metabolizing mutants of cytochrome CYP102A1 from Bacillus megaterium (CYP BM3) have been described, and from the onset, computational tools were integrated in the rationalization and design of novel mutants [49,71,72]. Two mutants of CYPBM3 were experimentally shown to catalyze the stereospecific hydroxylation of α-ionones [73].…”

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

View full text Add to dashboard Cite

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.

show abstract

“…Whereas wild-type (WT) BM3 only accepts long fatty acids as substrates, many successful protein engineering studies have been reported over the last years that report mutants of BM3 able to hydroxylate drugs and drug-like compounds with high activity and with regio-and/or stereo-selectivity [11][12][13][14][15][16][17][18][19][20][21]. However, rationally predicting novel mutants with enhanced selectivity and/or activity is still difficult especially when considering the flexibility of their active site and because they can typically bind a variety of substrates in multiple binding poses [18,[22][23][24][25][26]. In the present combined experimental and computational study we report and study the biocatalytic activity for (regioselective) 5′ methyl-hydroxylation of FLX by CYP BM3 mutants M11, M11 L437E and M11 L437A.…”

Section: Introductionmentioning

confidence: 99%

A combined computational and experimental study on selective flucloxacillin hydroxylation by cytochrome P450 BM3 variants

Luirink

Dekker

Capoferri

et al. 2018

Journal of Inorganic Biochemistry

View full text Add to dashboard Cite

The 5'-hydroxymethyl metabolite of the penicillin based antibiotic flucloxacillin (FLX) is considered to be involved in bile duct damage occurring in a small number of patients. Because 5'-hydroxymethyl FLX is difficult to obtain by organic synthesis, biosynthesis using highly active and regioselective biocatalysts would be an alternative approach. By screening an in-house library of Cytochrome P450 (CYP) BM3 mutants, mutant M11 L437E was identified as a regioselective enzyme with relatively high activity in production of 5'-hydroxymethyl FLX as was confirmed by mass spectrometry and NMR. In contrast, incubation of M11 L437E and other mutants with oxacillin (OX, which differs from FLX by a lack of aromatic halogens) resulted in formation of two metabolites. In addition to 5'-hydroxymethyl OX we identified a product resulting from aromatic hydroxylation. In silico studies of both FLX and OX with three CYP BM3 mutants revealed substrate binding poses allowing for 5'-methyl hydroxylation, as well as binding poses with the aromatic moiety in the vicinity of the heme iron for which the corresponding product of aromatic hydroxylation was not observed for FLX. Supported by the (differences in) experimentally determined ratios of product formation for OX hydroxylation by M11 and its L437A variant and M11 L437E, Molecular Dynamics simulations suggest that the preference of mutant M11 L437E to bind FLX in its catalytically active pose over the other binding orientation contributes to its biocatalytic activity, highlighting the benefit of studying effects of active-site mutations on possible alternative enzyme-substrate binding poses in protein engineering.

show abstract

The Role of Protein Plasticity in Computational Rationalization Studies on Regioselectivity in Testosterone Hydroxylation by Cytochrome P450 BM3 Mutants

Cited by 11 publications

References 15 publications

Prediction of Sites of Metabolism of CYP3A4 Substrates Utilizing Docking-Derived Geometric Features

Prediction of Sites of Metabolism of CYP3A4 Substrates Utilizing Docking-Derived Geometric Features

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

A combined computational and experimental study on selective flucloxacillin hydroxylation by cytochrome P450 BM3 variants

Contact Info

Product

Resources

About