Functional Analysis of the Region Linking the N-Terminal Transmembrane Anchor and the Catalytic Domain of Cytochrome P450 2C2

The linker region connecting the N-terminal signal anchor and the cytoplasmic catalytic domain of P450 2C2 contains two segments with distinct sequence properties: a Gly-rich and a Pro-rich region. The activities and spectral properties were determined for both Pro- and Gly-rich region mutants expressed in COS-1 cells, bacteria, and insect cells. In COS-1 cells, mutations at Pro 30 and Pro33 in the Pro-rich region dramatically reduced activity, suggesting that a PXXP motif may be important for the formation or activity of a functional P450, and further that this sequence might have a helical structure with a repeat of three, as in the left-handed polyproline II helix. Substitution of Pro30 and Pro33 with Ala resulted in a reduced P450 and increased P420 for the mutants expressed in bacteria or insect cells which correlated with the decreased activity in COS-1 cells. These data suggest the Pro-rich region is critical for the folding of P450. Substitution of the Gly residues in the Gly-rich region with Ala or Pro or the entire sequence from 22 to 28 with Ala did not reduce laurate hydroxylase activity of the proteins expressed in COS-1 cells. Deletion of residues 22-28 or substitution with valine inactivated the protein. Substitution of two Ala resulted in loss of activity in COS-1 cells, but activity increased progressively with substitution of 3 or 4 Ala to activities similar to wild-type. Lengthening the linker from 2 to 7 Ala resulted in progressively increased P450 which corresponded with decreased inactive P420 for the mutants expressed in bacteria or insect cells. Substitution of 7 Val resulted in only the P420 form of the protein and deletion of 22-28 resulted in neither P450 nor P420 forms. The activities per nmole P450 were similar for wild type and the mutants with 2 to 7 Ala substituted. These data are consistent with a role for the Gly-rich region as a linker which facilitates the folding of P450 into a functional protein, but is not required for the activity of the folded protein.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Molecular and Integrative Physiology

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2015

Author dc:creator

• Chen, Ci-Di

Contributors dc:contributor

• Byron Kemper

• Biology, Molecular

Language dc:language

eng

Identifier

(MiAaPQ)AAI9834662

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/87260