scholarly journals Riboflavin Directly Mediates the Dealkylation by Microbial Cytochrome P450 Monooxygeneses

2019 ◽  
Author(s):  
Chengchang Zhang ◽  
Meiling Lu ◽  
Lin Lin ◽  
Zhangjian Huang ◽  
Rongguang Zhang ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Cytochrome P450 ◽  
Biochemical Analysis ◽  
Site Directed Mutagenesis ◽  
Dynamics Simulation ◽  
Cytochrome P450 Monooxygenases ◽  
Electron Transfer Mechanism ◽  
X Ray Crystallography ◽  
P450 Monooxygenases ◽  
P450 Enzyme

ABSTRACTAs a vast repertoire of enzymes in nature, microbial cytochrome P450 monooxygenases require an activated form of flavin as a cofactor for the catalytic activity. Riboflavin is the precursor of FAD and FMN that serve as indispensable cofactors for flavoenzymes. In contrast to previous notion, here we describe the identification of an electron transfer process directly mediated by riboflavin for the N-dealkylation by microbial P450 monooxygenases. The electron relay from NADPH to riboflavin and then via activated oxygen to heme was proposed based on the combination of X-ray crystallography, molecular modeling and molecular dynamics simulation, site-directed mutagenesis and biochemical analysis of representative microbial P450 monooxygenases. This study provides new insights into the electron transfer mechanism in microbial P450 enzyme catalysis and likely in plants and mammals.

scholarly journals Conversion of viridicatic acid to crustosic acid by cytochrome P450 enzyme-catalysed hydroxylation and spontaneous cyclisation

2021 ◽  
Author(s):  
Jenny Zhou ◽  
Shu-Ming Li
Keyword(s):  
Cytochrome P450 ◽  
Genetic Manipulation ◽  
Selection Marker ◽  
Cytochrome P450 Monooxygenases ◽  
Cytochrome P450 Enzyme ◽  
Oxidation Reactions ◽  
Functional Identification ◽  
Marker Recycling ◽  
P450 Monooxygenases ◽  
P450 Enzyme

Abstract Cytochrome P450 monooxygenases (P450s) are considered nature’s most versatile catalysts and play a crucial role in regio- and stereoselective oxidation reactions on a broad range of organic molecules. The oxyfunctionalisation of unactivated carbon-hydrogen (C-H) bonds, in particular, represents a key step in the biosynthesis of many natural products as it provides substrates with increased reactivity for tailoring reactions. In this study, we investigated the function of the P450 enzyme TraB in the terrestric acid biosynthetic pathway. We firstly deleted the gene coding for the DNA repair subunit protein Ku70 by using split marker-based deletion plasmids for convenient recycling of the selection marker to improve gene targeting in Penicillium crustosum. Hereby, we reduced ectopic DNA integration and facilitated genetic manipulation in P. crustosum. Afterward, gene deletion in the Δku70 mutant of the native producer P. crustosum and heterologous expression in Aspergillus nidulans with precursor feeding proved the involvement of TraB in the formation of crustosic acid by catalysing the essential hydroxylation reaction of viridicatic acid. Key points •Deletion of Ku70 by using split marker approach for selection marker recycling. •Functional identification of the cytochrome P450 enzyme TraB. •Fulfilling the reaction steps in the terrestric acid biosynthesis.

Determination of organophosphate resistance status and mechanism in Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae) from Turkey / Türkiye’deki Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae)’nin organofosfat direnç durumunun ve mekanizmasının belirlenmesi

2015 ◽  
Vol 40 (5) ◽  
Author(s):  
Sakine Ugurlu Karaağaç ◽  
Metin Konuş
Keyword(s):  
Cytochrome P450 ◽  
Biochemical Analysis ◽  
Sitophilus Zeamais ◽  
Test Method ◽  
Cytochrome P450 Monooxygenases ◽  
Susceptible Population ◽  
Organophosphate Resistance ◽  
P450 Monooxygenases ◽  
Pirimiphos Methyl ◽  
Glutathione S Transferases

AbstractObjective: The objectives of this study were to determine resistance status to malathion and pirimiphos- methyl insecticides and to make biochemical analysis of resistance mechanism(s) developed to these insecticides in Sitophilus zeamais (S. zeamais) populations, collected from two different locations in Turkey. Two organophosphate insecticides, malathion and pirimiphos- methyl, were examined by bioassay using a discriminating dosage technique with impregnated filter papers. Mortality percentages were determined at the discriminating doses of these insecticides. In addition, esterase, glutathione S-transferase, and cytochrome P450 monooxygenase activities were also determined in this study in order to analyze detoxification mechanism(s) of tested insecticides in S. zeamais.Methods: Bioassay experiments of malathion and pirimiphos- methyl insecticides in S. zeamais populations were performed according to the IRAC susceptibility test method No:006. Furthermore, enzyme activities of esterases, cytochrome P450 monooxygenases, and glutathione S-transferases were determined by using biochemical assays.Results: The bioassay results of malathion and pirimiphos- methyl showed that only Kırıkkale population of S. zeamais has resistance to both malathion and pirimiphos- methyl insecticides. However, no resistance was detected to malathion and pirimiphos-methyl insecticides in Samsun population of S. zeamais. Additionally, biochemical analysis displayed that while CYP450-PNOD activities showed an increase only in Kırıkkale population (3.0-fold), EST-PNPA activities showed an increase only in Samsun population (1.3-fold). Finally, GST-CDNB activities increased both in Kırıkkale (1.4-fold) and Samsun (2.2- fold) populations of S. zeamais compared to susceptible population.Conclusion: Consequently, cytochrome P450 monooxygenases and glutathione S-transferases seem to play a role in organophosphate resistance in Kırıkkale population of S. zeamais from Turkey.

NON-MICHAELIS-MENTEN KINETICS IN CYTOCHROME P450-CATALYZED REACTIONS

2005 ◽  
Vol 45 (1) ◽  
pp. 291-310 ◽  
Author(s):  
William M. Atkins
Keyword(s):  
Cytochrome P450 ◽  
Drug Metabolism ◽  
Active Sites ◽  
Cytochrome P450 Monooxygenases ◽  
X Ray Crystallography ◽  
Xenobiotic Detoxification ◽  
P450 Monooxygenases ◽  
New Information ◽  
Multiple Ligands ◽  
Catalyzed Reactions

The cytochrome P450 monooxygenases (CYPs) are the dominant enzyme system responsible for xenobiotic detoxification and drug metabolism. Several CYP isoforms exhibit non-Michaelis-Menten, or “atypical,” steady state kinetic patterns. The allosteric kinetics confound prediction of drug metabolism and drug-drug interactions, and they challenge the theoretical paradigms of allosterism. Both homotropic and heterotropic ligand effects are now widely documented. It is becoming apparent that multiple ligands can simultaneously bind within the active sites of individual CYPs, and the kinetic parameters change with ligand occupancy. In fact, the functional effect of any specific ligand as an activator or inhibitor can be substrate dependent. Divergent approaches, including kinetic modeling and X-ray crystallography, are providing new information about how multiple ligand binding yields complex CYP kinetics.

scholarly journals Regio- and Stereospecific Hydroxylation of Various Steroids at the 16α Position of the D Ring by the Streptomyces griseus Cytochrome P450 CYP154C3

2013 ◽  
Vol 80 (4) ◽  
pp. 1371-1379 ◽  
Author(s):  
Takuya Makino ◽  
Yohei Katsuyama ◽  
Toshihiko Otomatsu ◽  
Norihiko Misawa ◽  
Yasuo Ohnishi
Keyword(s):  
Cytochrome P450 ◽  
High Resolution Mass Spectrometry ◽  
Streptomyces Griseus ◽  
Industrial Applications ◽  
Cytochrome P450 Monooxygenases ◽  
Direct Oxidation ◽  
Content Type ◽  
P450 Monooxygenases ◽  
P450 Enzyme ◽  
Equilibrium Dissociation

ABSTRACTCytochrome P450 monooxygenases (P450s), which constitute a superfamily of heme-containing proteins, catalyze the direct oxidation of a variety of compounds in a regio- and stereospecific manner; therefore, they are promising catalysts for use in the oxyfunctionalization of chemicals. In the course of our comprehensive substrate screening for all 27 putative P450s encoded by theStreptomyces griseusgenome, we found thatEscherichia colicells producing anS. griseusP450 (CYP154C3), which was fused C terminally with the P450 reductase domain (RED) of a self-sufficient P450 fromRhodococcussp., could transform various steroids (testosterone, progesterone, Δ4-androstene-3,17-dione, adrenosterone, 1,4-androstadiene-3,17-dione, dehydroepiandrosterone, 4-pregnane-3,11,20-trione, and deoxycorticosterone) into their 16α-hydroxy derivatives as determined by nuclear magnetic resonance and high-resolution mass spectrometry analyses. The purified CYP154C3, which was not fused with RED, also catalyzed the regio- and stereospecific hydroxylation of these steroids at the same position with the aid of ferredoxin and ferredoxin reductase from spinach. The apparent equilibrium dissociation constant (Kd) values of the binding between CYP154C3 and these steroids were less than 8 μM as determined by the heme spectral change, indicating that CYP154C3 strongly binds to these steroids. Furthermore, kinetic parameters of the CYP154C3-catalyzed hydroxylation of Δ4-androstene-3,17-dione were determined (Km, 31.9 ± 9.1 μM;kcat, 181 ± 4.5 s−1). We concluded that CYP154C3 is a steroid D-ring 16α-specific hydroxylase which has considerable potential for industrial applications. This is the first detailed enzymatic characterization of a P450 enzyme that has a steroid D-ring 16α-specific hydroxylation activity.

Characterization of cytochrome P450-monooxygenases of Chinese hamsters with respect to aflatoxin B1 activation

Toxicology ◽  
1994 ◽  
Vol 93 (2-3) ◽  
pp. 165-173 ◽  
Author(s):  
Morio Fukuhara ◽  
Eric Antignac ◽  
Naomi Fukusen ◽  
Kazue Kato ◽  
Masanobu Kimura
Keyword(s):  
Cytochrome P450 ◽  
Aflatoxin B1 ◽  
Cytochrome P450 Monooxygenases ◽  
P450 Monooxygenases ◽  
Chinese Hamsters

scholarly journals Specificity and mechanism of carbohydrate demethylation by cytochrome P450 monooxygenases

2018 ◽  
Vol 475 (23) ◽  
pp. 3875-3886 ◽  
Author(s):  
Craig S. Robb ◽  
Lukas Reisky ◽  
Uwe T. Bornscheuer ◽  
Jan-Hendrik Hehemann
Keyword(s):  
Cytochrome P450 ◽  
Active Site ◽  
Substrate Recognition ◽  
High Energy ◽  
Cytochrome P450 Monooxygenases ◽  
P450 Monooxygenases ◽  
High Energy Barrier ◽  
Molecular Determinants ◽  
Cytochrome P450 Family ◽  
Carbohydrate Ligand

Degradation of carbohydrates by bacteria represents a key step in energy metabolism that can be inhibited by methylated sugars. Removal of methyl groups, which is critical for further processing, poses a biocatalytic challenge because enzymes need to overcome a high energy barrier. Our structural and computational analysis revealed how a member of the cytochrome P450 family evolved to oxidize a carbohydrate ligand. Using structural biology, we ascertained the molecular determinants of substrate specificity and revealed a highly specialized active site complementary to the substrate chemistry. Invariance of the residues involved in substrate recognition across the subfamily suggests that they are critical for enzyme function and when mutated, the enzyme lost substrate recognition. The structure of a carbohydrate-active P450 adds mechanistic insight into monooxygenase action on a methylated monosaccharide and reveals the broad conservation of the active site machinery across the subfamily.

scholarly journals Biotechnological Methods of Sulfoxidation: Yesterday, Today, Tomorrow

Catalysts ◽  
2018 ◽  
Vol 8 (12) ◽  
pp. 624 ◽  
Author(s):  
Wanda Mączka ◽  
Katarzyna Wińska ◽  
Małgorzata Grabarczyk
Keyword(s):  
Cytochrome P450 ◽  
Chemical Industry ◽  
Functional Materials ◽  
Cytochrome P450 Monooxygenases ◽  
Whole Cells ◽  
Historical Significance ◽  
P450 Monooxygenases ◽  
Bacteria And Fungi ◽  
Positive Results ◽  
Huge Variety

The production of chiral sulphoxides is an important part of the chemical industry since they have been used not only as pharmaceuticals and pesticides, but also as catalysts or functional materials. The main purpose of this review is to present biotechnological methods for the oxidation of sulfides. The work consists of two parts. In the first part, examples of biosyntransformation of prochiral sulfides using whole cells of bacteria and fungi are discussed. They have more historical significance due to the low predictability of positive results in relation to the workload. In the second part, the main enzymes responsible for sulfoxidation have been characterized such as chloroperoxidase, dioxygenases, cytochrome flavin-dependent monooxygenases, and P450 monooxygenases. Particular emphasis has been placed on the huge variety of cytochrome P450 monooxygenases, and flavin-dependent monooxygenases, which allows for pure sulfoxides enantiomers effectively to be obtained. In the summary, further directions of research on the optimization of enzymatic sulfoxidation are indicated.

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