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J Biol Chem, Vol. 275, Issue 4, 2545-2553, January 28, 2000
From the Department of Molecular and Experimental Medicine, The
Scripps Research Institute, La Jolla, California 92037
Deletion of the N-terminal membrane-spanning
domain from microsomal P450s 2C5 and 2C3 generates the enzymes, 2C5dH
and 2C3dH, that exhibit a salt-dependent association with
membranes indicating that they retain a monofacial membrane interaction
domain. The two proteins are tetramers and dimers, respectively, in
high salt buffers, and only 2C5dH requires phospholipids to
reconstitute fully the catalytic activity of the enzyme. Amino acid
residues derived from P450 2C3dH between residues 201 and 210 were
substituted for the corresponding residues in P450 2C5 to identify
those that would diminish the membrane interaction, the phospholipid
dependence of catalysis, and aggregation of 2C5dH. Each of four
substitutions, N202H, I207L, S209G, and S210T, diminished the
aggregation of P450 2C5dH and produced a monomeric enzyme. The N202H
and I207L mutations also diminished the stimulation of catalytic
activity by phospholipid and reduced the binding of P450 2C5dH to
phospholipid vesicles. The modified enzymes exhibit rates of
progesterone 21-hydroxylation that are similar to that of P450
2C5dH. These conditionally membrane-bound P450s with improved
solubility in high salt buffers are suitable for crystallization and
structural determination by x-ray diffraction studies.
Engineering Microsomal Cytochrome P450 2C5 to Be a Soluble,
Monomeric Enzyme
MUTATIONS THAT ALTER AGGREGATION, PHOSPHOLIPID DEPENDENCE OF
CATALYSIS, AND MEMBRANE BINDING*
*
This work was supported by United States Public Health
Service Grant GM31001.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: Biochemistry, NX-4,
The Scripps Research Institute, 10550 North Torrey Pines Rd., La Jolla,
CA 92037. Tel.: 858-784-7918; Fax: 858-784-7981; E-mail: johnson@
scripps.edu.
Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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