Abstract
Cytochrome P450 is anchored to the endoplasmic reticulum membrane by an N-terminal transmembrane sequence with the catalytic domain facing the cytoplasmic side. Within the peptide sequence linking these two domains is a highly conserved proline-rich region. In cytochrome P450 2C2, this region has the sequence 30PPGPTPFP37. To examine the structural requirements at these proline residues, each proline was replaced with alanine, glycine, valine, or an acidic amino acid, and the activities of the mutated proteins were determined in transfected COS-1 cells. Lauric acid 1omega-hydroxylase activities of Pro30 and Pro33 mutants were less than 10% of wild type for each substitution except for alanine, which was 25-30%. In striking contrast, substitutions at Pro31, including an acidic residue, did not substantially alter activity. At positions 35 and 37, acidic amino acid substitutions reduced activity to less than 10% of wild type while substitution of the other three amino acids had little effect. The tolerance of substitutions of charged residues at Pro31 suggests that the side chain at this position is exposed to a polar environment; conversely, the reduced activity with charged substitutions, but not with uncharged substitutions at positions 35 and 37, suggests that these residues are exposed to a hydrophobic environment, presumably within the folded protein. The loss of activity with substitutions at Pro30 and Pro33 implies that the motif PXXP is important for the formation of a functional cytochrome P450 and that this sequence might have a helical structure with a repeat of three, as in the left-handed poly-L-proline II helix. Insertion of alanine between positions 29 and 30 did not substantially affect activity, but insertions between either 33 and 34 or 37 and 38 resulted in activity less than 25% of wild type. These data indicate that the position of PXXP, relative to the sequence flanking it on the C-terminal side, may be important for its function.
Highlights
Cytochrome P450 (P450)1 refers to a superfamily of proteins that metabolize a wide variety of endogenous and xenobiotic compounds and are located in the mitochondria or microsomes of mammalian cells [1, 2]
The results demonstrate different structural requirements at specific Pro residue positions and suggest that a PXXP motif may be important for the formation of a functional P450 2C2
Insertions of Ala between residues 33 and 34 or 37 and 38 reduced activity to less than 25% of the activity of wild type, similar to the Ala substitutions at Pro30 and Pro33. These results suggest that the position of the PXXP motif relative to flanking sequence on the C-terminal side is important for the formation of functional P450 2C2, while the position relative to N-terminal sequences is not
Summary
P450, cytochrome P450; ER, endoplasmic reticulum; PAGE, polyacrylamide gel electrophoresis; PPII, poly(Lproline) II; SH3, Src homology 3; HIV, human immunodeficiency virus. The sequence PPGP is highly conserved with the identical sequence present in family 2 P450s The function of these Pro residues is not known, but they could be important for a turn out of the membrane, which determines the orientation of the protein relative to the membrane, for the folding or incorporation of heme in newly synthesized P450, or for the stability or catalytic activity of the mature protein. Substitutions of Ala for the first Pro in the PPGP sequence of P450 2C11, or for combinations of two or three Pro residues including the first or last Pro in each case, resulted in mutant proteins that were expressed in yeast but that did not exhibit the characteristic difference spectrum at 450 nm [13] These data indicated that the mutant proteins did not fold to form a functional P450 and supported the conclusion that this region was important for the folding of the protein or the incorporation of the heme moiety. The results demonstrate different structural requirements at specific Pro residue positions and suggest that a PXXP motif may be important for the formation of a functional P450 2C2
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