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Structural importance of residue 334 in the stability of cytochromes P450 2B6 and 2B11
Author(s) -
Talakad Jyothi C,
Wilderman P. Ross,
Davydov Dmitri R,
Kumar Santosh,
Halpert James R
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.967.14
Subject(s) - chemistry , residue (chemistry) , heme , serine , mutant , enzyme , amino acid , alanine , biochemistry , mutagenesis , proline , site directed mutagenesis , stereochemistry , gene
Human P450 2B6 and canine P450 2B11 play an important role in xenobiotic metabolism, bioremediation research, and activation of anti‐cancer prodrugs. Thorough biochemical and biophysical characterization of the enzymes has been impeded by relatively low stability compared to the extensively studied rat 2B1 and rabbit 2B4. Comparison of the amino acid sequences of N‐terminal truncated and C‐terminal His‐tagged constructs revealed seven sites that are conserved between the more stable 2B1 and 2B4 but different from those found in the less stable 2B6 and 2B11. Site‐directed mutagenesis at these positions showed that P 334 → Ser substitution increased heterologous expression levels and thermal stability in both 2B6 and 2B11. The mechanism of this effect was explored with pressure‐perturbation spectroscopy. Compressibility of the heme pocket in the four P450 2B enzymes and their P334S (2B6 and 2B11) or S334P (2B1 and 2B4) mutants was assessed from the pressure induced displacement of the Soret band. The variants of all four enzymes containing proline at position 334 are characterized by lower compressibility than their more stable serine 334 counterparts. Therefore, the stabilizing effect of P334S is associated with increased conformational flexibility in the region of the heme pocket. Mutation of the non‐active site residue P 334 → Ser could find applications in drug metabolism and gene therapy for cancer treatment.