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J. Biol. Chem., Vol. 278, Issue 19, 17178-17184, May 9, 2003
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From the Department of Pharmacology and Toxicology, University of
Texas Medical Branch, Galveston, Texas 77555-1031
The regioselectivity for progesterone
hydroxylation by cytochrome P450 2B1 was re-engineered based on the
x-ray crystal structure of cytochrome P450 2C5. 2B1 is a high
Km progesterone 16
A Rational Approach to Re-engineer Cytochrome P450 2B1
Regioselectivity Based on the Crystal Structure of Cytochrome P450
2C5*
,
-hydroxylase, whereas 2C5 is a
low Km progesterone 21-hydroxylase. Initially, nine
individual 2B1 active-site residues were changed to the corresponding
2C5 residues, and the mutants were purified from an Escherichia
coli expression system and assayed for progesterone
hydroxylation. At 150 µM progesterone, I114A, F297G, and
V363L showed 5-15% of the 21-hydroxylase activity of 2C5, whereas
F206V showed high activity for an unknown product and a 13-fold
decrease in Km. Therefore, a quadruple mutant,
I114A/F206V/F297G/V363L (Q), was constructed that showed 60% of 2C5
progesterone 21-hydroxylase activity and 57% regioselectivity. Based
on their 2C5-like testosterone hydroxylation profiles, S294D and I477F
alone and in combination were added to the quadruple mutant. All three
mutants showed enhanced regioselectivity (70%) for progesterone
21-hydroxylation, whereas only Q/I477F had a higher
kcat. Finally, the remaining three single
mutants, V103I, V367L, and G478V, were added to Q/I477F and
Q/S294D/I477F, yielding seven additional multiple mutants. Among these,
Q/V103I/S294D/I477F showed the highest kcat
(3-fold higher than that of 2C5) and 80% regioselectivity for
progesterone 21-hydroxylation. Docking of progesterone into a
three-dimensional model of this mutant indicated that 21-hydroxylation
is favored. In conclusion, a systematic approach to convert P450
regioselectivity across subfamilies suggests that active-site residues
are mainly responsible for regioselectivity differences between
2B1 and 2C5 and validates the reliability of 2B1 models based on the
crystal structure of 2C5.
*
This work was supported by National Institutes of Health
Grant ES03619 and Center Grant ES06676.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Pharmacology
and Toxicology, University of Texas Medical Branch, 301 University
Blvd., Galveston, TX 77555-1031. Tel.: 409-772-9677; Fax: 409-772-9642;
E-mail: sakumar@utmb.edu.
Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
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