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All microsomal P450s have a proline-rich sequence (PR) in the amino-terminal region that is needed for proper folding [Kusano, K., Sakaguchi, M., Kagawa, N., Waterman, M.R. and Omura, T. (2001) J. Biochem., 129, 259-269]. There 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 P450s, we expressed human P450c27 (CYP27) and bovine P450scc (CYP11A1) in an Escherichia coli heterologous expression system, and found that in each one specific proline residue is important for correct folding. Deletions from the amino-terminus further indicated the importance of the PR for the expression of a spectrally normal P450c27. Essentially the same results were obtained with two soluble microbial P450s, P450cam (CYP101) and P450nor, in each of which a PR is important for proper folding. We conclude that in all P450s (mitochondrial, microbial and microsomal P450s), a proline-rich sequence located in the amino-terminal region is important for proper folding. Furthermore, 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.