Characterization of the consequence of a novel Glu-380 to Asp mutation by expression of functional P450c21 in Escherichia coli

Nai Chi Hsu, Victor M. Guzov, Li Chung Hsu, Bon Chu Chung

研究成果: Contribution to journalArticle査読

6 被引用数 (Scopus)

抄録

P450c21 catalyzes an important step in steroid synthesis. Its deficiency leads to symptoms of steroid imbalance. To obtain enough P450c21 for structure and function studies, we developed a method to express P450c21 in Escherichia coli. The 5'-region of the human P450c21 cDNA was modified to ensure efficient translation and the C terminus of the protein was extended with four His residues for easy purification. Mutant proteins with substitutions at residues 172 and 281 exhibited decreased enzymatic activities similar to those found in mammalian cells. One new mutation changing Glu-380 to Asp (D380) caused 3-fold reduction in enzymatic activity. The amount of apoprotein production detected by immunoblotting and the affinity of the mutant protein towards substrate as measured by K(m) were normal. The defect lies in the decreased ability of the apoprotein to bind heme, which was measured by CO difference and substrate-binding spectra. The D380 mutant protein had 3-fold reduction in peak heights in both spectra. This reduced heme binding resulted in 3-fold lower enzymatic activity. Copyright (C) 1999 Elsevier Science B.V.

本文言語英語
ページ(範囲)95-102
ページ数8
ジャーナルBiochimica et Biophysica Acta - Protein Structure and Molecular Enzymology
1430
1
DOI
出版ステータス出版済み - 2 10 1999

All Science Journal Classification (ASJC) codes

  • 生物理学
  • 構造生物学
  • 生化学
  • 分子生物学

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