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

P450c21 catalyzes an important step in steroid synthesis. Its deficiency le ads to symptoms of steroid imbalance. To obtain enough P450c21 for structur e and function studies, we developed a method to express P450c21 in Escheri chia coli. The 5'-region of the human P450c21 cDNA was modified to ensure e fficient translation and the C terminus of the protein was extended with fo ur His residues for easy purification. Mutant proteins with substitutions a t residues 172 and 281 exhibited decreased enzymatic activities similar to those found in mammalian cells. One new mutation changing Glu-380 to Asp (D 380) caused 3-fold reduction in enzymatic activity. The amount of apoprotei n production detected by immunoblotting and the affinity of the mutant prot ein towards substrate as measured by K-m were normal. The defect lies in th e decreased ability of the apoprotein to bind heme, which was measured by C O difference and substrate-binding spectra. The D380 mutant protein had 3-f old reduction in peak heights in both spectra. This reduced heme binding re sulted in 3-fold lower enzymatic activity. (C) 1999 Elsevier Science B.V. A ll rights reserved.