3BE0
The Role of Asn 242 in P450cin
Summary for 3BE0
| Entry DOI | 10.2210/pdb3be0/pdb |
| Related | 1T2B 3BDZ |
| Descriptor | P450cin, PROTOPORPHYRIN IX CONTAINING FE, 1,3,3-TRIMETHYL-2-OXABICYCLO[2.2.2]OCTANE, ... (4 entities in total) |
| Functional Keywords | protein substrate complex, unknown function |
| Biological source | Citrobacter braakii |
| Total number of polymer chains | 2 |
| Total formula weight | 90768.79 |
| Authors | Meharenna, Y.T.,Poulos, T.L. (deposition date: 2007-11-15, release date: 2008-02-12, Last modification date: 2023-11-01) |
| Primary citation | Meharenna, Y.T.,Slessor, K.E.,Cavaignac, S.M.,Poulos, T.L.,De Voss, J.J. The critical role of substrate-protein hydrogen bonding in the control of regioselective hydroxylation in p450cin J.Biol.Chem., 283:10804-10812, 2008 Cited by PubMed Abstract: Cytochrome P450cin (CYP176A1) is a bacterial P450 isolated from Citrobacter braakii that catalyzes the hydroxylation of cineole to (S)-6beta-hydroxycineole. This initiates the biodegradation of cineole, enabling C. braakii to live on cineole as its sole source of carbon and energy. P450cin lacks the almost universally conserved threonine residue believed to be involved in dioxygen activation and instead contains an asparagine at this position (Asn-242). To investigate the role of Asn-242 in P450cin catalysis, it was converted to alanine, and the resultant mutant was characterized. The characteristic CO-bound spectrum and spectrally determined K(D) for substrate binding were unchanged in the mutant. The x-ray crystal structures of the substrate-free and -bound N242A mutant were determined and show that the only significant change is in a reorientation of the substrate such that (R)-6alpha-hydroxycineole should be a major product. Molecular dynamics simulations of both wild type and mutant are consistent with the change in regio- and stereoselectivity predicted from the crystal structure. The mutation has only a modest effect on enzyme activity and on the diversion of the NADPH-reducing equivalent toward unproductive peroxide formation. Product profile analysis shows that (R)-6alpha-hydroxycineole is the main product, which is consistent with the crystal structure. These results demonstrate that Asn-242 is not a functional replacement for the conserved threonine in other P450s but, rather, is critical in controlling regioselective substrate oxidation. PubMed: 18270198DOI: 10.1074/jbc.M709722200 PDB entries with the same primary citation |
| Experimental method | X-RAY DIFFRACTION (3.05 Å) |
Structure validation
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