Identification and analysis of conserved sequence motifs in cytochrome P450 family 2

Functional and structural role of a motif187 RFDYKD 192 in CYP2B enzymes

Numan Oezguen, Santosh Kumar, Aditya Hindupur, Werner Braun, B. K. Muralidhara, James R. Halpert

Research output: Contribution to journalArticle

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Abstract

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 to 187RFDYKD 192 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, kcat, Km, and/or k cat/Km for 7-MFC and 7-EFC and 3- to 6-fold decreases in differential inhibition (IC50,BIF/IC50'4-CPI). Subsequently, these mutants displayed 5-12° C decreases in thermal stability (Tm) 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 IC50,BIF/IC50'4-CPI compared with 2B1dH. Analysis of long range residue-residue interactions in the CYP2B4 crystal structures indicated strong hydrogen bonds involving Glu149-Asn 177-Arg187-Tyr190 and Asp192- Val194, which were significantly reduced/abolished by the Arg 187 → Ala and Asp192 → Ala substitutions, respectively.

Original languageEnglish (US)
Pages (from-to)21808-21816
Number of pages9
JournalJournal of Biological Chemistry
Volume283
Issue number31
DOIs
StatePublished - Aug 1 2008

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bifonazole
Conserved Sequence
Cytochrome P-450 Enzyme System
Inhibitory Concentration 50
Thermodynamic stability
Enzymes
Hot Temperature
Substrates
Testosterone
Conservation
Hydrogen bonds
Substitution reactions
Substrate Specificity
Crystal structure
Hydrogen
Cats
Mutation
Temperature
cytochrome P-450 CYP2B4 (rabbit)
coumarin 7

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Cite this

Identification and analysis of conserved sequence motifs in cytochrome P450 family 2 : Functional and structural role of a motif187 RFDYKD 192 in CYP2B enzymes. / Oezguen, Numan; Kumar, Santosh; Hindupur, Aditya; Braun, Werner; Muralidhara, B. K.; Halpert, James R.

In: Journal of Biological Chemistry, Vol. 283, No. 31, 01.08.2008, p. 21808-21816.

Research output: Contribution to journalArticle

Oezguen, Numan ; Kumar, Santosh ; Hindupur, Aditya ; Braun, Werner ; Muralidhara, B. K. ; Halpert, James R. / Identification and analysis of conserved sequence motifs in cytochrome P450 family 2 : Functional and structural role of a motif187 RFDYKD 192 in CYP2B enzymes. In: Journal of Biological Chemistry. 2008 ; Vol. 283, No. 31. pp. 21808-21816.
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abstract = "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 to 187RFDYKD 192 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, kcat, Km, and/or k cat/Km for 7-MFC and 7-EFC and 3- to 6-fold decreases in differential inhibition (IC50,BIF/IC50'4-CPI). Subsequently, these mutants displayed 5-12° C decreases in thermal stability (Tm) 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 IC50,BIF/IC50'4-CPI compared with 2B1dH. Analysis of long range residue-residue interactions in the CYP2B4 crystal structures indicated strong hydrogen bonds involving Glu149-Asn 177-Arg187-Tyr190 and Asp192- Val194, which were significantly reduced/abolished by the Arg 187 → Ala and Asp192 → Ala substitutions, respectively.",
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T2 - Functional and structural role of a motif187 RFDYKD 192 in CYP2B enzymes

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AU - Kumar, Santosh

AU - Hindupur, Aditya

AU - Braun, Werner

AU - Muralidhara, B. K.

AU - Halpert, James R.

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N2 - 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 to 187RFDYKD 192 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, kcat, Km, and/or k cat/Km for 7-MFC and 7-EFC and 3- to 6-fold decreases in differential inhibition (IC50,BIF/IC50'4-CPI). Subsequently, these mutants displayed 5-12° C decreases in thermal stability (Tm) 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 IC50,BIF/IC50'4-CPI compared with 2B1dH. Analysis of long range residue-residue interactions in the CYP2B4 crystal structures indicated strong hydrogen bonds involving Glu149-Asn 177-Arg187-Tyr190 and Asp192- Val194, which were significantly reduced/abolished by the Arg 187 → Ala and Asp192 → Ala substitutions, respectively.

AB - 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 to 187RFDYKD 192 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, kcat, Km, and/or k cat/Km for 7-MFC and 7-EFC and 3- to 6-fold decreases in differential inhibition (IC50,BIF/IC50'4-CPI). Subsequently, these mutants displayed 5-12° C decreases in thermal stability (Tm) 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 IC50,BIF/IC50'4-CPI compared with 2B1dH. Analysis of long range residue-residue interactions in the CYP2B4 crystal structures indicated strong hydrogen bonds involving Glu149-Asn 177-Arg187-Tyr190 and Asp192- Val194, which were significantly reduced/abolished by the Arg 187 → Ala and Asp192 → Ala substitutions, respectively.

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