Importance of a proline-rich sequence in the amino-terminal region for correct folding of mitochondrial and soluble microbial P450s

All microsomal P450s have a proline-rich sequence (PR) in the amino-termina l region that is needed for proper folding [Kusano, K., Sakaguchi, M., Kaga wa, N., Waterman, M.R. and Omura, T. (2001) J. Biochem., 129, 259-269]. The re are also multiple proline residues near the amino-termini of the mature forms of all mitochondrial P450s and the amino-termini of soluble microbial P450s. To examine the functional significance of the PR in mitochondrial P 450s, we expressed human P450c27 (CYP27) and bovine P450scc (CYP11A1) in an Escherichia coli heterologous expression system, and found that in each on e specific proline residue is important for correct folding. Deletions from the amino-terminus further indicated the importance of the PR for the expr ession of a spectrally normal P450c27, Essentially the same results were ob tained with two soluble microbial P450s, P450cam (CYP101) and P450nor, in e ach of which a PR is important for proper folding. We conclude that in all P450s (mitochondrial, microbial and microsomal P450s), a proline-rich seque nce located in the amino-terminal region is important for proper folding. F urthermore, we predict that the importance of the PR in P450 folding is to reduce the tendency of the polypeptide to misfold prior to heme binding.