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Catalytic site mutagenesis of cytochrome P450 6B1v1

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Title: Catalytic site mutagenesis of cytochrome P450 6B1v1
Author(s): Pookanjanatavip, Manee
Doctoral Committee Chair(s): Sligar, Stephen G.
Department / Program: Biochemistry
Discipline: Biochemistry
Degree Granting Institution: University of Illinois at Urbana-Champaign
Degree: Ph.D.
Genre: Dissertation
Subject(s): Biology, Molecular Chemistry, Biochemistry
Abstract: Cytochromes P-450 form a large superfamily of heme-containing monooxygenases that are found in species ranging from bacteria to insects, plants, and mammals. In insects, P-450s have several functional roles, including growth, development, feeding, resistance to pesticides, and tolerance to plant toxins. Cytochrome P-450 6B1v1 (CYP6B1v1) is a membrane-bound P-450 derived from larvae of the black swallowtail butterfly, Papilio polyxenes, in midgut microsomes. It predominantly involves the detoxification or metabolism of xanthotoxin, a linear furanocoumarin, with very little capability to metabolize angelicin, an angular furanocoumarin. Both furanocoumarins are naturally occurring secondary plant metabolites with the highly phototoxic property.To understand substrate specificity and catalysis of CYP6B1v1, the spatial relationships of the amino acids at the catalytic site must be established by solving the three-dimensional structure. Such a structure is not available for the membrane-bound microsomal P-450s. In this thesis, the mouse P-450 2a-5 (Cyp2a-5) with the similar substrate recognition to insect P-450 6B1v1 (CYP6B1v1) is however used as the model. The xanthotoxin, a substrate of CYP6B1v1, can be a substrate and a competitive inhibitor of Cyp2a-5. Although CYP6B1v1 cannot metabolize coumarin, a substrate of Cyp2a-5, it can be competitively inhibited by coumarin. It indicates that the catalytic sites of CYP6B1v1 and Cyp2a-5 are structurally similar.In Cyp2a-5, three residues, V117, F209, and M365, have been shown to be critical for substrate specificity and catalysis. On the basis of the alignment of Cyp2a-5 and CYP6B1v1, three residues, V117, F209, and M365, in Cyp2a-5 correspond to F116, H204, and V368 in CYP6B1v1 respectively. The question is posed whether these 3 residues, F116, H204, and V368, are critical for specificity and catalysis of xanthotoxin and angelicin in CYP6B1v1. Using site-directed mutagenesis and determining kinetic constants, K$\rm\sb{m}$ and V$\sb{\max},$ the results suggest that F116 does appear to play an important role in xanthotoxin binding in catalytic site, H204 may affect xanthotoxin catalysis but not binding, and V368 may not affect xanthotoxin recognition significantly. With angelicin metabolism, these 3 residues may not contribute to angelicin recognition significantly. The mutations of these residues do not appear to increase the specificity of angelicin. In general, the understanding of catalytic site of CYP6B1v1 is of interest in the rational design of inhibitors or pesticides including redesigning P-450 specificities.
Issue Date: 1996
Type: Text
Language: English
URI: http://hdl.handle.net/2142/20144
ISBN: 9780591088250
Rights Information: Copyright 1996 Pookanjanatavip, Manee
Date Available in IDEALS: 2011-05-07
Identifier in Online Catalog: AAI9702640
OCLC Identifier: (UMI)AAI9702640
 

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