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

Cytochrome P450 Enzyme ◽

Oxidation Reactions ◽

Functional Identification ◽

Marker Recycling ◽

P450 Monooxygenases ◽

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.

Cytochrome P450 enzyme mimics in ‘peroxo-shunt’ oxidation reactions – a kinetic and mechanistic approach

BioInorganic Reaction Mechanisms ◽

10.1515/birm.2011.001 ◽

2011 ◽

Vol 7 (1-4) ◽

Author(s):

Alicja Franke ◽

Christoph Fertinger ◽

Rudi van Eldik

Keyword(s):

Cytochrome P450 ◽

Cytochrome P450 Enzyme ◽

Enzyme Mimics ◽

Oxidation Reactions ◽

Mechanistic Approach ◽

Riboflavin Directly Mediates the Dealkylation by Microbial Cytochrome P450 Monooxygeneses

10.1101/801928 ◽

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 ◽

Electron Transfer Mechanism ◽

X Ray Crystallography ◽

P450 Monooxygenases ◽

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.

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

Applied and Environmental Microbiology ◽

10.1128/aem.03504-13 ◽

2013 ◽

Vol 80 (4) ◽

pp. 1371-1379 ◽

Cited By ~ 28

Author(s):

Takuya Makino ◽

Yohei Katsuyama ◽

Toshihiko Otomatsu ◽

Norihiko Misawa ◽

Yasuo Ohnishi

Keyword(s):

Cytochrome P450 ◽

High Resolution Mass Spectrometry ◽

Streptomyces Griseus ◽

Industrial Applications ◽

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.

Mechanism of Oxidation Reactions Catalyzed by Cytochrome P450 Enzyme

ChemInform ◽

10.1002/chin.200446299 ◽

2004 ◽

Vol 35 (46) ◽

Cited By ~ 1

Author(s):

Bernard Meunier ◽

Samuel P. de Visser ◽

Sason Shaik

Keyword(s):

Cytochrome P450 ◽

Cytochrome P450 Enzyme ◽

Oxidation Reactions ◽

P450 Enzyme ◽

Mechanism Of Oxidation

Active site diversification of P450cam with indole generates catalysts for benzylic oxidation reactions

Beilstein Journal of Organic Chemistry ◽

10.3762/bjoc.11.186 ◽

2015 ◽

Vol 11 ◽

pp. 1713-1720 ◽

Cited By ~ 13

Author(s):

Paul P Kelly ◽

Anja Eichler ◽

Susanne Herter ◽

David C Kranz ◽

Nicholas J Turner ◽

...

Keyword(s):

Amino Acids ◽

Cytochrome P450 ◽

Active Site ◽

Mutant Library ◽

Oxidation Reactions ◽

P450 Monooxygenases ◽

Highly Sensitive ◽

Fusion Enzymes ◽

Benzylic Oxidation

Cytochrome P450 monooxygenases are useful biocatalysts for C–H activation, and there is a need to expand the range of these enzymes beyond what is naturally available. A panel of 93 variants of active self-sufficient P450cam[Tyr96Phe]-RhFRed fusion enzymes with a broad diversity in active site amino acids was developed by screening a large mutant library of 16,500 clones using a simple, highly sensitive colony-based colorimetric screen against indole. These mutants showed distinct fingerprints of activity not only when screened in oxidations of substituted indoles but also for unrelated oxidations such as benzylic hydroxylations.

A Bioorganometallic Chemistry Overview: From Cytochrome P450 Enzyme Metabolism of Organotin Compounds to Organorhodium-Hydroxytamoxifen Complexes with Potential Anti-Cancer Properties; A 37 Year Perspective at the Interface of Organometallic Chemistry and Biology

Australian Journal of Chemistry ◽

10.1071/ch10239 ◽

2010 ◽

Vol 63 (11) ◽

pp. 1505 ◽

Cited By ~ 32

Author(s):

Richard H. Fish

Keyword(s):

Cytochrome P450 ◽

Organotin Compounds ◽

Cytochrome P450 Enzyme ◽

Oxidation Reactions ◽

Tandem Catalysis ◽

Cytochrome P450 Enzymes ◽

Exciting Field ◽

Bioorganometallic Chemistry ◽

Hydride Reduction ◽

A 37 year perspective on bioorganometallic chemistry studies, which included metabolism of organotin compounds with cytochrome P450 enzymes, and their biomimics; reactions of organorhodium aqua complexes with nucleobases, nucleosides, and nucleotides; supramolecular organorhodium-nucleobase complexes as hosts for aromatic amino acid and aromatic carboxylic acid guests; regioselective reduction of NAD+ biomimics with an organorhodium hydride; tandem catalysis of an organorhodium hydride reduction to provide a 1,4-NADH biomimic for horse liver dehydrogenase stereoselective reduction of achiral ketones to chiral alcohols, and oxidation reactions with cytochrome P450 enzymes; and organorhodium-hydroxytamoxifen pharmaceuticals, will be presented. Each of these areas of bioorganometallic chemistry will be briefly discussed in this personal synopsis of the new, important, and exciting field of bioorganometallic chemistry, and its impact on metal-based drug research.