HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 283, Issue 31, 21808-21816, August 1, 2008

This Article Full Text Full Text (PDF) Supplemental Data All Versions of this Article: 283/31/21808    most recent M708582200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Oezguen, N. Articles by Halpert, J. R. Search for Related Content PubMed PubMed Citation Articles by Oezguen, N. Articles by Halpert, J. R. Social Bookmarking                      What's this?

Identification and Analysis of Conserved Sequence Motifs in Cytochrome P450 Family 2

FUNCTIONAL AND STRUCTURAL ROLE OF A MOTIF ¹⁸⁷RFDYKD¹⁹² IN CYP2B ENZYMES^*

Numan Oezguen¹, Santosh Kumar¹², Aditya Hindupur, Werner Braun, B. K. Muralidhara^¶, and James R. Halpert

From the Skaggs School of Pharmacy & Pharmaceutical Sciences, University of California San Diego, La Jolla, California 92093, the Department of Biochemistry and Molecular Biology and The Sealy Center for Structural Biology and Molecular Biophysics, University of Texas Medical Branch, Galveston, Texas 77555-1031, and ^¶Pfizer Inc., PhRD-Global Biologics, Chesterfield, Missouri 63017

Using a multiple alignment of 175 cytochrome P450 (CYP) family 2 sequences, 20 conserved sequence motifs (CSMs) were identified with the program PCPMer. Functional importance of the CSM in CYP2B enzymes was assessed from available data on site-directed mutants and genetic variants. These analyses suggested an important role of the CSM 8, which corresponds to¹⁸⁷RFDYKD¹⁹² in CYP2B4. Further analysis showed that residues 187, 188, 190, and 192 have a very high rank order of conservation compared with 189 and 191. Therefore, eight mutants (R187A, R187K, F188A, D189A, Y190A, K191A, D192A, and a negative control K186A) were made in an N-terminal truncated and modified form of CYP2B4 with an internal mutation, which is termed 2B4dH/H226Y. Function was examined with the substrates 7-methoxy-4-(trifluoromethyl)coumarin (7-MFC), 7-ethoxy-4-(trifluoromethyl)coumarin (7-EFC), 7-benzyloxy-4-(trifluoromethyl)coumarin (7-BFC), and testosterone and with the inhibitors 4-(4-chlorophenyl)imidazole (4-CPI) and bifonazole (BIF). Compared with the template and K186A, the mutants R187A, R187K, F188A, Y190A, and D192A showed 2-fold altered substrate specificity, k_cat, K_m, and/or k_cat/K_m for 7-MFC and 7-EFC and 3- to 6-fold decreases in differential inhibition (IC_50,BIF/IC_50,4-CPI). Subsequently, these mutants displayed 5-12 °C decreases in thermal stability (T_m) and 2-8 °C decreases in catalytic tolerance to temperature (T₅₀) compared with the template and K186A. Furthermore, when R187A and D192A were introduced in CYP2B1dH, the P450 expression and thermal stability were decreased. In addition, R187A showed increased activity with 7-EFC and decreased IC_50,BIF/IC_50,4-CPI compared with 2B1dH. Analysis of long range residue-residue interactions in the CYP2B4 crystal structures indicated strong hydrogen bonds involving Glu¹⁴⁹-Asn¹⁷⁷-Arg¹⁸⁷-Tyr¹⁹⁰ and Asp¹⁹²-Val¹⁹⁴, which were significantly-reduced/abolished by the Arg¹⁸⁷Ala and Asp¹⁹²Alasubstitutions, respectively.

Received for publication, October 16, 2007 , and in revised form, May 20, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grant ES03619 and Center Grant ES06676 (to J. R. H.) and by NIH Grant AI 064913 (to W. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables S1-S3.

¹ Both authors contributed equally to this work.

² To whom correspondence should be addressed: Skaggs School of Pharmacy & Pharmaceutical Sciences, University of California San Diego, 9500 Gilman Drive, Rm. 2231, La Jolla, CA 92093-0703. Tel.: 858-822-7804; Fax: 858-246-0089; E-mail: s8kumar{at}ucsd.edu.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				S. Kumar, H. Qiu, N. Oezguen, H. Herlyn, J. R. Halpert, and L. Wojnowski Ligand Diversity of Human and Chimpanzee CYP3A4: Activation of Human CYP3A4 by Lithocholic Acid Results from Positive Selection Drug Metab. Dispos., June 1, 2009; 37(6): 1328 - 1333. [Abstract] [Full Text] [PDF]

				J. C. Talakad, S. Kumar, and J. R. Halpert Decreased Susceptibility of the Cytochrome P450 2B6 Variant K262R to Inhibition by Several Clinically Important Drugs Drug Metab. Dispos., March 1, 2009; 37(3): 644 - 650. [Abstract] [Full Text] [PDF]