scholarly journals Catalytic Mechanism of Cytochrome P450 for 5′-Hydroxylation of Nicotine: Fundamental Reaction Pathways and Stereoselectivity

2011 ◽  
Vol 133 (19) ◽  
pp. 7416-7427 ◽  
Author(s):  
Dongmei Li ◽  
Xiaoqin Huang ◽  
Keli Han ◽  
Chang-Guo Zhan
Keyword(s):  
Cytochrome P450 ◽  
Catalytic Mechanism ◽  
Reaction Pathways

scholarly journals Characterization of the structure and interactions of P450 BM3 using hybrid mass spectrometry approaches

2020 ◽  
Vol 295 (22) ◽  
pp. 7595-7607 ◽  
Author(s):  
Laura N. Jeffreys ◽  
Kamila J. Pacholarz ◽  
Linus O. Johannissen ◽  
Hazel M. Girvan ◽  
Perdita E. Barran ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Cytochrome P450 ◽  
Bound States ◽  
Catalytic Mechanism ◽  
Catalytic Domain ◽  
Full Length ◽  
P450 Bm3 ◽  
Interaction Sites ◽  
Domain Interactions ◽  
Hdx Ms

The cytochrome P450 monooxygenase P450 BM3 (BM3) is a biotechnologically important and versatile enzyme capable of producing important compounds such as the medical drugs pravastatin and artemether, and the steroid hormone testosterone. BM3 is a natural fusion enzyme comprising two major domains: a cytochrome P450 (heme-binding) catalytic domain and a NADPH-cytochrome P450 reductase (CPR) domain containing FAD and FMN cofactors in distinct domains of the CPR. A crystal structure of full-length BM3 enzyme is not available in its monomeric or catalytically active dimeric state. In this study, we provide detailed insights into the protein-protein interactions that occur between domains in the BM3 enzyme and characterize molecular interactions within the BM3 dimer by using several hybrid mass spectrometry (MS) techniques, namely native ion mobility MS (IM-MS), collision-induced unfolding (CIU), and hydrogen-deuterium exchange MS (HDX-MS). These methods enable us to probe the structure, stoichiometry, and domain interactions in the ∼240 kDa BM3 dimeric complex. We obtained high-sequence coverage (88–99%) in the HDX-MS experiments for full-length BM3 and its component domains in both the ligand-free and ligand-bound states. We identified important protein interaction sites, in addition to sites corresponding to heme-CPR domain interactions at the dimeric interface. These findings bring us closer to understanding the structure and catalytic mechanism of P450 BM3.

scholarly journals 3P100 X-ray crystal structures of cytochrome P450 StaP which produces skeleton of an anticancer drug suggest unusual catalytic mechanism of P450(Hemeproteins,Poster Presentations)

2007 ◽  
Vol 47 (supplement) ◽  
pp. S228
Author(s):  
Masatomo Makino ◽  
Shingo Nagao ◽  
Hiroshi Sugimoto ◽  
Shumpei Asamizu ◽  
Hiroyasu Onaka ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Crystal Structures ◽  
Anticancer Drug ◽  
Catalytic Mechanism ◽  
X Ray ◽  
Poster Presentations

Catalytic Mechanism of Cytochrome P450 2D6 for 4-Hydroxylation of Aripiprazole: A QM/MM Study

2013 ◽  
Vol 31 (9) ◽  
pp. 1219-1227 ◽  
Author(s):  
Rongwei Shi ◽  
Weihua Li ◽  
Guixia Liu ◽  
Yun Tang
Keyword(s):  
Cytochrome P450 ◽  
Catalytic Mechanism ◽  
Cytochrome P450 2D6 ◽  
P450 2D6

The catalytic mechanism of cytochrome P450 BM3 involves a 6 Å movement of the bound substrate on reduction

1996 ◽  
Vol 3 (5) ◽  
pp. 414-417 ◽  
Author(s):  
Sandeep Modi ◽  
Michael J. Sutcliffe ◽  
William U. Primrose ◽  
Lu-Yun Lian ◽  
Gordon C.K. Roberts
Keyword(s):  
Cytochrome P450 ◽  
Catalytic Mechanism ◽  
P450 Bm3 ◽  
Cytochrome P450 Bm3

scholarly journals Two distinct catalytic pathways for GH43 xylanolytic enzymes unveiled by X-ray and QM/MM simulations

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Mariana A. B. Morais ◽  
Joan Coines ◽  
Mariane N. Domingues ◽  
Renan A. S. Pirolla ◽  
Celisa C. C. Tonoli ◽  
...  
Keyword(s):  
Active Site ◽  
Catalytic Mechanism ◽  
Open Architecture ◽  
Reaction Pathways ◽  
Main Reaction ◽  
Glycoside Hydrolase Family ◽  
X Ray ◽  
Glycoside Hydrolase Family 43 ◽  
Xylanolytic Enzymes ◽  
Hydrolase Family

AbstractXylanolytic enzymes from glycoside hydrolase family 43 (GH43) are involved in the breakdown of hemicellulose, the second most abundant carbohydrate in plants. Here, we kinetically and mechanistically describe the non-reducing-end xylose-releasing exo-oligoxylanase activity and report the crystal structure of a native GH43 Michaelis complex with its substrate prior to hydrolysis. Two distinct calcium-stabilized conformations of the active site xylosyl unit are found, suggesting two alternative catalytic routes. These results are confirmed by QM/MM simulations that unveil the complete hydrolysis mechanism and identify two possible reaction pathways, involving different transition state conformations for the cleavage of xylooligosaccharides. Such catalytic conformational promiscuity in glycosidases is related to the open architecture of the active site and thus might be extended to other exo-acting enzymes. These findings expand the current general model of catalytic mechanism of glycosidases, a main reaction in nature, and impact on our understanding about their interaction with substrates and inhibitors.

Characterization of the kaurene oxidase CYP701A3, a multifunctional cytochrome P450 from gibberellin biosynthesis

2010 ◽  
Vol 431 (3) ◽  
pp. 337-347 ◽  
Author(s):  
Dana Morrone ◽  
Xiaoming Chen ◽  
Robert M. Coates ◽  
Reuben J. Peters
Keyword(s):  
Cytochrome P450 ◽  
Catalytic Mechanism ◽  
Recombinant Expression ◽  
Ring Structure ◽  
Gibberellin Biosynthesis ◽  
Reaction Sequence ◽  
Acid Moiety ◽  
Enzymatic Mechanism ◽  
Rate Limiting

KO (kaurene oxidase) is a multifunctional cytochrome P450 catalysing three sequential oxidations in gibberellin phytohormone biosynthesis. These serve to transform the C4α methyl of the ent-kaurene olefin intermediate into the carboxylic acid moiety of ent-kauren-19-oic acid. To investigate the unknown catalytic mechanism and properties of KO, we have engineered the corresponding CYP701A3 from Arabidopsis thaliana (AtKO) for functional recombinant expression in Escherichia coli, involving use of a fully codon-optimized construct, along with additional N-terminal deletion and modification. This recombinant AtKO (rAtKO) was used to carry out 18O2 labelling studies with ent-kaurene, and the intermediates ent-kaurenol and ent-kaurenal, to investigate the multifunctional reaction sequence; revealing catalysis of three hydroxylation reactions, which further requires dehydration at some stage. Accordingly, following initial hydroxylation, ent-kaurenol must then be further hydroxylated to a gem-diol intermediate, and our data indicate that the subsequent reactions proceed via dehydration of the gem-diol to ent-kaurenal, followed by an additional hydroxylation to directly form ent-kaurenoic acid. Kinetic analysis indicates that these intermediates are all retained in the active site during the course of the reaction series, with the first hydroxylation being rate-limiting. In addition, investigation of alternative substrates demonstrated that ent-beyerene, which differs in ring structure distal to the C4α methyl, is only hydroxylated by rAtKO, indicating the importance of the exact tetracyclic ring structure of kaurane for multifunctional KO activity. Thus the results of the present study clarify the reaction sequence and enzymatic mechanism of KO, as well as substrate features critical for the catalysed multiple reaction sequence.

scholarly journals Catalytic Mechanism of Aromatic Nitration by Cytochrome P450 TxtE: Involvement of a Ferric-Peroxynitrite Intermediate

2020 ◽  
Vol 142 (37) ◽  
pp. 15764-15779 ◽  
Author(s):  
Savvas Louka ◽  
Sarah M. Barry ◽  
Derren J. Heyes ◽  
M. Qadri E. Mubarak ◽  
Hafiz Saqib Ali ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Catalytic Mechanism ◽  
Aromatic Nitration
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