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

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)95-102
Number of pages8
JournalBiochimica et Biophysica Acta - Protein Structure and Molecular Enzymology
Volume1430
Issue number1
DOIs
Publication statusPublished - Feb 10 1999

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Mutant Proteins
Escherichia coli
Apoproteins
Heme
Mutation
Steroids
Substrates
Carbon Monoxide
Protein C
Immunoblotting
Purification
Substitution reactions
Complementary DNA
Cells
Defects
Proteins

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Structural Biology
  • Biochemistry
  • Molecular Biology

Cite this

Characterization of the consequence of a novel Glu-380 to Asp mutation by expression of functional P450c21 in Escherichia coli. / Hsu, Nai Chi; Guzov, Victor M.; Hsu, Li Chung; Chung, Bon Chu.

In: Biochimica et Biophysica Acta - Protein Structure and Molecular Enzymology, Vol. 1430, No. 1, 10.02.1999, p. 95-102.

Research output: Contribution to journalArticle

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