A functional proline switch in cytochrome P450cam.
The two-protein complex between putidaredoxin (Pdx) and cytochrome P450(cam) (CYP101) is the catalytically competent species for camphor hydroxylation by CYP101. We detected a conformational change in CYP101 upon binding of Pdx that reorients bound camphor appropriately for hydroxylation. Experimental evidence shows that binding of Pdx converts a single X-proline amide bond in CYP101 from trans or distorted trans to cis. Mutation of proline 89 to isoleucine yields a mixture of both bound camphor orientations, that seen in Pdx-free and that seen in Pdx-bound CYP101. A mutation in CYP101 that destabilizes the cis conformer of the Ile 88-Pro 89 amide bond results in weaker binding of Pdx. This work provides direct experimental evidence for involvement of X-proline isomerization in enzyme function.
Department of Chemistry, Brandeis University, 415 South Street, Waltham, MA 02454-9110, USA.
Bo
B
OuYang
Susan Sondej
SS
Pochapsky
Marina
M
Dang
Thomas C
TC
Pochapsky
D001665:Binding Sites; D019475:Camphor 5-Monooxygenase / Q000737:chemistry* / Q000235:genetics; D005288:Ferredoxins / Q000737:chemistry*; D007536:Isomerism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D011392:Proline / Q000737:chemistry*; D011487:Protein Conformation
D005288:Ferredoxins; C100137:putidaredoxin; D011392:Proline; D019475:Camphor 5-Monooxygenase