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 language | English |
|---|---|
| Pages (from-to) | 95-102 |
| Number of pages | 8 |
| Journal | Biochimica et Biophysica Acta - Protein Structure and Molecular Enzymology |
| Volume | 1430 |
| Issue number | 1 |
| DOIs | |
| Publication status | Published - Feb 10 1999 |
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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 journal › Article
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TY - JOUR
T1 - Characterization of the consequence of a novel Glu-380 to Asp mutation by expression of functional P450c21 in Escherichia coli
AU - Hsu, Nai Chi
AU - Guzov, Victor M.
AU - Hsu, Li Chung
AU - Chung, Bon Chu
PY - 1999/2/10
Y1 - 1999/2/10
N2 - 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.
AB - 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.
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UR - http://www.scopus.com/inward/citedby.url?scp=0033056213&partnerID=8YFLogxK
U2 - 10.1016/S0167-4838(98)00271-4
DO - 10.1016/S0167-4838(98)00271-4
M3 - Article
C2 - 10082937
AN - SCOPUS:0033056213
VL - 1430
SP - 95
EP - 102
JO - Biochimica et Biophysica Acta - Proteins and Proteomics
JF - Biochimica et Biophysica Acta - Proteins and Proteomics
SN - 1570-9639
IS - 1
ER -