Role of C-terminal sequence of cytochrome P450scc in folding and functional activity.
To elucidate the role of Arg472 and C-terminal sequence of the mature form of cytochrome P450scc, a mitochondrial cytochrome P450, in the present work we have performed sequential removal of the C-terminal amino acid residues of the hemeprotein and evaluated their functional role in folding and catalysis. The removal of 2, 4, 7, or 9 amino acid residues (cytochrome P450scc mutants Delta2, Delta4, Delta7, and Delta9) does not significantly affect the physicochemical properties of the truncated forms of cytochrome P450scc, but results in significant increase in the expression level of the hemeprotein in Escherichia coli (Delta4 cytochrome P450scc mutant). However, removal of 10 C-terminal amino acid residues (Delta10 cytochrome P450scc) of mature form of cytochrome P450scc (replacement of codon for Arg472 for stop-codon) is followed by loss of the ability for correct folding in E. coli. Based on these data, it is concluded that the C-terminal amino acid residues of cytochrome P450scc (DeltaArg472-Ala481) play an important role in correct recombinant protein folding and heme binding by cytochrome P450scc during its expression in E. coli, while folding of mitochondrial cytochrome P450scc during its heterologous expression in bacterial cells is more similar to the folding of prokaryotic soluble cytochrome P450's than to microsomal cytochrome P450's.
Strushkevich
N V
NV
Institute of Bioorganic Chemistry, National Academy of Sciences of Belarus, Minsk 220141, Belarus.
Harnastai
I N
IN
Lepesheva
G I
GI
Usanov
S A
SA
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000737:chemistry; D002417:Cattle; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000737:chemistry* / Q000378:metabolism; D002942:Circular Dichroism; D004926:Escherichia coli / Q000201:enzymology; D016297:Mutagenesis, Site-Directed; D017510:Protein Folding; D025941:Protein Interaction Mapping; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D011994:Recombinant Proteins; D001120:Arginine; D002786:Cholesterol Side-Chain Cleavage Enzyme