Elucidation of Amino Acid Residues Critical for Unique Activities of Rabbit Cytochrome P450 2B5 Using Hybrid Enzymes and Reciprocal Site-Directed Mutagenesis with Rabbit Cytochrome P450 2B4

Szklarz, Grazyna D.; He, Yeping; Kedzie, Karen M.; Halpert, James R.; Burnett, Vicki L.

doi:10.1006/abbi.1996.0127

Cited by 51 publications

(35 citation statements)

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“…In earlier studies, I363V in 2B4 shows a > 3-fold enhanced activity with androstenedione and V363L in P450 2B1dH shows a 2-fold enhanced activity with testosterone compared with the respective wild-type [7,25,26]. In contrast, V363A in 2B1 shows a > 4-fold lower androstenedione hydroxylation than 2B1 [26].…”

Section: Steady-state Kinetic Analysis Of Testosterone Hydroxylationmentioning

confidence: 85%

“…In addition, several other studies with 2B enzymes such as 2B1 have shown that decreasing the size of residue at 363 favors 15α-hydroxylation of androstenedione [26]. V367A in P450 2B4 shows altered stereoselectivity of androstenedione hydroxylation [25], and I477F in P450 2B1dH shows altered testosterone hydroxylase stereoselectivity [7].…”

Section: Stereo-and Regioselectivity Of Testosterone Hydroxylationmentioning

confidence: 89%

“…Residue 363 has been shown to be involved in differential substrate specificity and strict stereo-and regioselectivity in several P450 2B enzymes [1,7,[23][24][25][26][27]. For example, a smaller residue at position 363 in P450 2B4 and P450 2B5 prefers androstenedione 15α-hydroxylation, whereas a larger one prefers androstenedione 16α-hydroxylation [25].…”

Section: Stereo-and Regioselectivity Of Testosterone Hydroxylationmentioning

confidence: 99%

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Investigation of the role of cytochrome P450 2B4 active site residues in substrate metabolism based on crystal structures of the ligand-bound enzyme

Hernandez

Kumar

Liu

et al. 2006

Archives of Biochemistry and Biophysics

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Based on the x-ray crystal structures of 4-(4-chlorophenyl)imidazole (4-CPI)-and bifonazole (BIF)-bound P450 2B4, eight active site mutants at six positions were created in an N-terminal modified construct termed 2B4dH and characterized for enzyme inhibition and catalysis. I363A showed a > 4-fold decrease in differential inhibition by BIF and 4-CPI (IC 50,BIF /IC 50,4-CPI ). F296A, T302A, I363A, V367A, and V477A showed a ≥ 2-fold decreased k cat for 7-ethoxy-4-trifluoromethylcoumarin O-deethylation, whereas V367A and V477F showed an altered K m . T302A, V367L, and V477A showed a > 4-fold decrease in total testosterone hydroxylation, whereas I363A, V367A, and V477F showed altered stereo-and regioselectivity. Interestingly, I363A showed a ≥ 150-fold enhanced k cat /K m with testosterone, and yielded a new metabolite. Furthermore, testosterone docking into three-dimensional models of selected mutants based on the 4-CPI-bound structure suggested a re-positioning of residues 363 and 477 to yield products. In conclusion, our results suggest that the 4-CPI-bound 2B4dH/H226Y crystal structure is an appropriate model for predicting enzyme catalysis.

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Section: Steady-state Kinetic Analysis Of Testosterone Hydroxylationmentioning

confidence: 85%

Section: Stereo-and Regioselectivity Of Testosterone Hydroxylationmentioning

confidence: 89%

Section: Stereo-and Regioselectivity Of Testosterone Hydroxylationmentioning

confidence: 99%

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Investigation of the role of cytochrome P450 2B4 active site residues in substrate metabolism based on crystal structures of the ligand-bound enzyme

Hernandez

Kumar

Liu

et al. 2006

Archives of Biochemistry and Biophysics

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“…Through the alignment of sequences from known members of the CYP2 family, Gotoh (10) assigned possible substrate recognition sites (SRS), which have formed the basis of studies by other groups. Based on those types of analyses, several groups have used site-directed mutagenesis to explore the role of functional residues (11)(12)(13)). An alternative technique utilizes the catalytic ability of the enzyme to convert a bound substrate to a reactive product capable of modifying the active site protein or heme (14,15).…”

Section: Introductionmentioning

confidence: 99%

CYP2E1 active site residues in substrate recognition sequence 5 identified by photoaffinity labeling and homology modeling

Collom¹,

Jamakhandi²,

Tackett³

et al. 2007

Archives of Biochemistry and Biophysics

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Despite its biological importance, our knowledge of active site structure and relevance of critical amino acids in CYP2E1 catalytic processes remain limited. In this study, we identified CYP2E1 active site residues using photoaffinity labeling with 7-azido-4-methylcoumarin (AzMC) coupled with a CYP2E1 homology model. In the absence of light, AzMC was an effective competitor against substrate p-nitrophenol oxidation by CYP2E1. Photoactivation of AzMC led to a concentrationdependent loss in CYP2E1 activity and structural integrity resulting from the modification of both heme and protein. The photolabeling reaction degraded heme and produced a possible heme adduct. Probe incorporation into the protein occurred at multiple sites within substrate recognition sequence 5 (SRS-5). Based on a CYP2E1 homology model, we hypothesize AzMC labels SRS-5 residues, Leu363, Val364 and Leu368, in the active site. In addition, we propose a series of phenylalanines, especially Phe106, mediate contacts with the coumarin.

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“…Many of these residues belong to five of the six substraterecognition sites (SRSs) for P450 family 2 enzymes that were proposed on the basis of multiple sequence alignments and analogy with P450 101 (Gotoh, 1992). In the case of the P450 2B enzymes, residues 114, 363, and 367 account for many of the functional differences between rat 2B1 and 2B2 (Strobel and Halpert, 1997), rabbit 2B4 and 2B5 Szklarz et al, 1996), rat 2B1 and human 2B6 (Domanski et al, 1999), and rat 2B1 and canine 2B11 . These functional alterations can frequently be explained using three-dimensional homology models based on the structures of bacterial P450s (Szklarz and Halpert, 1997) or P450 2C5 .…”

mentioning

confidence: 99%

Activation of the Anticancer Prodrugs Cyclophosphamide and Ifosfamide: Identification of Cytochrome P450 2B Enzymes and Site-Specific Mutants with Improved Enzyme Kinetics

Chen¹,

Lin²,

Goss³

et al. 2004

Mol Pharmacol

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Cyclophosphamide (CPA) and ifosfamide (IFA) are oxazaphosphorine anticancer prodrugs metabolized by two alternative cytochrome P450 (P450) pathways, drug activation by 4-hydroxylation and drug inactivation by N-dechloroethylation, which generates the neurotoxic and nephrotoxic byproduct chloroacetaldehyde. CPA and IFA metabolism catalyzed by P450s 2B1, 2B4, 2B5, and seven site-specific 2B1 mutants was studied in a reconstituted Escherichia coli expression system to identify residues that contribute to the unique activities and substrate specificities of these enzymes. The catalytic efficiency of CPA 4-hydroxylation by rat P450 2B1 was 10-to 35-fold higher than that of rabbit P450 2B4 or 2B5. With IFA, ϳ50% of metabolism proceeded via N-dechloroethylation for 2B1 and 2B4, whereas CPA N-dechloroethylation corresponded to only ϳ3% of total metabolism (2B1) or was absent (2B4, 2B5). Improved catalytic efficiency of CPA and IFA 4-hydroxylation was obtained upon substitution of 2B1 Ile-114 by Val, and replacement of Val-363 by Leu or Ile selectively suppressed CPA N-dechloroethylation Ն90%. P450 2B1-V367A, containing the Ala replacement found in 2B5, exhibited only ϳ10% of wild-type 2B1 activity for both substrates. Canine P450 2B11, which has Val-114, Leu-363, and Val-367, was therefore predicted to be a regioselective CPA 4-hydroxylase with high catalytic efficiency. Indeed, P450 2B11 was 7-to 8-fold more active as a CPA and IFA 4-hydroxylase than 2B1, exhibited a highly desirable low K m (80 -160 M), and catalyzed no CPA N-dechloroethylation. These findings provide insight into the role of specific P450 2B residues in oxazaphosphorine metabolism and pave the way for gene therapeutic applications using P450 enzymes with improved catalytic activity toward these anticancer prodrug substrates.The oxazaphosphorine cyclophosphamide (CPA) and its structural isomer ifosfamide (IFA) are DNA-alkylating agents commonly used in cancer chemotherapy (Sladek, 1994). These anticancer agents are administered as prodrugs that are activated by a liver cytochrome P450-catalyzed 4-hydroxylation reaction that yields active, cytotoxic metabolites. Several liver-expressed P450 enzymes catalyze this activation reaction; the phenobarbital-inducible rat P450 enzyme 2B1 (Clarke and Waxman, 1989) and its human counterpart 2B6 (Chang et al., 1993;Huang et al., 2000) show particularly high CPA 4-hydroxylase activity compared with other P450 forms. The primary metabolite, 4-OH-CPA or 4-OH-IFA, equilibrates with the ring-open aldophosphamide and undergoes ␤-elimination to yield the therapeutically active, DNA cross-linking phosphoramide mustard and the byproduct acrolein (4-hydroxylation pathway). CPA and IFA are also subject to an alternative, P450-catalyzed side chain oxidation that generates therapeutically inactive N-dechloroethylated metabolites and the neurotoxic and nephrotoxic byproduct chloroacetaldehyde (CAA; N-dechloroethylation pathway) (Furlanut and Franceschi, 2003).In patients with cancer, CPA and IFA are primarily activated...

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Elucidation of Amino Acid Residues Critical for Unique Activities of Rabbit Cytochrome P450 2B5 Using Hybrid Enzymes and Reciprocal Site-Directed Mutagenesis with Rabbit Cytochrome P450 2B4

Cited by 51 publications

References 0 publications

Investigation of the role of cytochrome P450 2B4 active site residues in substrate metabolism based on crystal structures of the ligand-bound enzyme

Investigation of the role of cytochrome P450 2B4 active site residues in substrate metabolism based on crystal structures of the ligand-bound enzyme

CYP2E1 active site residues in substrate recognition sequence 5 identified by photoaffinity labeling and homology modeling

Activation of the Anticancer Prodrugs Cyclophosphamide and Ifosfamide: Identification of Cytochrome P450 2B Enzymes and Site-Specific Mutants with Improved Enzyme Kinetics

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