Characterization of stable human aromatase expressed in E. coli.

Kagawa N, Hori H, Waterman MR, Yoshioka S
Steroids. 2004 69 (4): 235-43

PMID: 15183689 · DOI:10.1016/j.steroids.2004.01.002

Aromatase (P450arom, CYP19) catalyzes the aromatization reaction that converts androgens to estrogens. Although human P450arom has been readily purified from placenta, its hydrophobic properties and instability has hampered detailed characterization. Utilizing a N-terminal replacement (MARQSFGRGKL, derived from CYP2C11), we successfully modified this unstable enzyme into stable forms. Based on a known polymorphism, we created two constructs, NmA264C and NmA264R having cysteine or arginine at position 264. The recombinant P450arom NmA264R was expressed in Escherichia coli (350-400 nmol/L culture) primarily by coexpression with molecular chaperones GroES/GroEL while NmA264C was expressed (240 nmol/L culture) only in the presence of chloramphenicol. Although NmA264C was recovered only in the membrane fraction, approximately 14% of NmA264R was recovered in the cytosolic fraction, suggesting that NmA264R is more hydrophilic than NmA264C. NmA264R was highly purified to the specific content 13.6 nmol P450/mg protein. Purified P450arom NmA264R converted androstenedione to estrone with Vmax 12.4 nmol/(min nmol) and Km) 0.26 microM, and testosterone to estradiol with Vmax 52.2 nmol/(min nmol) and Km 10.9 microM. Because of the increased stability of NmA264R, we could unambiguously determine properties of human P450arom by optical and electron paramagnetic resonance spectroscopy. The purified protein was a typical low-spin form, which was converted to a high-spin form when androstenedione was added. The rhombicity of substrate-bound forms was higher than that reported for other P450s, an interesting characteristic of human P450arom. The highly stable and active P450arom NmA264R sets the stope for detailed structure/function analyses of this important member of the P450 superfamily.

MeSH Terms (28)

Amino Acid Sequence Androstenedione Arginine Aromatase Aromatase Inhibitors Cell Membrane Chaperonin 10 Chaperonin 60 Chloramphenicol Cysteine Electron Spin Resonance Spectroscopy Enzyme Stability Escherichia coli Estradiol Estrone Fadrozole Gene Expression Humans Letrozole Molecular Sequence Data Nitriles Polymorphism, Genetic Protein Structure, Secondary Protein Synthesis Inhibitors Recombinant Proteins Solubility Testosterone Triazoles

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