PDBsum entry 1n6b

Oxidoreductase

PDB id: 1n6b

Contents

Protein chain

462 a.a. *

Ligands

SO4 ×2

HEM

DMZ

Waters ×118

* Residue conservation analysis

References listed in PDB file

Key reference

Title

Structure of a substrate complex of mammalian cytochrome p450 2c5 at 2.3 a resolution: evidence for multiple substrate binding modes.

Authors

M.R.Wester, E.F.Johnson, C.Marques-Soares, P.M.Dansette, D.Mansuy, C.D.Stout.

Ref.

Biochemistry, 2003, 42, 6370-6379. [DOI no: 10.1021/bi0273922]

PubMed id

12767218

Abstract

The structure of rabbit microsomal cytochrome P450 2C5/3LVdH complexed with a substrate, 4-methyl-N-methyl-N-(2-phenyl-2H-pyrazol-3-yl)benzenesulfonamide (DMZ), was determined by X-ray crystallography to 2.3 A resolution. Substrate docking studies and electron density maps indicate that DMZ binds to the enzyme in two antiparallel orientations of the long axis of the substrate. One orientation places the principal site of hydroxylation, the 4-methyl group, 4.4 A from the heme Fe, whereas the alternate conformation positions the second, infrequent site of hydroxylation at >5.9 A from the heme Fe. Comparison of this structure to that obtained previously for the enzyme indicates that the protein closes around the substrate and prevents open access of water from bulk solvent to the heme Fe. This reflects a approximately 1.5 A movement of the F and G helices relative to helix I. The present structure provides a complete model for the protein from residues 27-488 and defines two new helices F' and G'. The G' helix is likely to contribute to interactions of the enzyme with membranes. The relatively large active site, as compared to the volume occupied by the substrate, and the flexibility of the enzyme are likely to underlie the capacity of drug-metabolizing enzymes to metabolize structurally diverse substrates of different sizes.

Secondary reference #1

Title

Mammalian microsomal cytochrome p450 monooxygenase: structural adaptations for membrane binding and functional diversity.

Authors

P.A.Williams, J.Cosme, V.Sridhar, E.F.Johnson, D.E.Mcree.

Ref.

Mol Cell, 2000, 5, 121-131. [DOI no: 10.1016/S1097-2765(00)80408-6]

PubMed id

10678174

Figure 1.
Figure 1. Electron Density MapThe final σ[A] ([39]) weighted 2F[o] − F[c] electron density map contoured at 1 σ and superimposed on the final model. Helix I runs horizontally across the center of the picture. The potential interaction between the side chain of Thr-298 and the main chain carbonyl of Ala-294 can be seen just above the heme.

Figure 3.
Figure 3. Conservation of the Heme Binding SiteThe heme is sandwiched between helix I on the distal side and helix L on the proximal side of the protein. The side chain S[χ] of Cys-432 (data not shown) is an axial ligand to the heme iron and is found immediately upstream of helix L. These features are spatially conserved with other P450s as shown by the superposition of P450 BM3 (yellow) on the structure of 2C5/3LVdH (cyan). In addition, helix C is well conserved in space. The two molecules were superimposed by an rms fit of C[α] atoms in helix L, the β bulge, and the central portion of helix I. The β strand 1–4 and the immediate upstream region located across the distal surface of the heme from the I helix exhibit extensive divergence, with an rms deviation of 3.3 Å between C[α] atoms.

The above figures are reproduced from the cited reference with permission from Cell Press

Secondary reference #2

Title

Engineering microsomal cytochrome p450 2c5 to be a soluble, Monomeric enzyme. Mutations that alter aggregation, Phospholipid dependence of catalysis, And membrane binding.

Authors

J.Cosme, E.F.Johnson.

Ref.

J Biol Chem, 2000, 275, 2545-2553. [DOI no: 10.1074/jbc.275.4.2545]

PubMed id

10644712

Figure 1.
Fig. 1. Sequence alignment of P450s 2C5 and 2C3 (or 2C3v) with the sequence of P450 BM3 in the helix F to helix G region. Only differences from the sequence of P450 2C5 are shown for P450 2C3. The secondary structure shown for P450 BM3 below the alignment was determined experimentally by Ravichandran et al. (29). The secondary structure of P450 2C5 predicted by the Self-optimized Prediction Method from Alignment according to Geourjon and DeLeage (5) is depicted above the alignment. The region of the sequences of P450s 2C3 and 2C5 used for the construction of reciprocal chimeras is boxed. Residue numbers for 2C5 and BM3 are shown immediately above and below the sequence alignment, respectively.

Figure 6.
Fig. 6. Estimation of the oligomerization of P450s 2C3dH, 2C5dH, and 2C5/3LVdH by size exclusion chromatography. The scale indicates the elapsed time in minutes from injection of the sample. The retention times (min) for 2C5/3LVdH, 2C3dH, and 2C5dH are 30.1, 28.3, and 24.6 min, respectively. The traces were obtained by monitoring the absorption of visible light at 417 nm by the eluant.

The above figures are reproduced from the cited reference with permission from the ASBMB

Author's comment:  The results of the size exclusion chromatography indicated that substitution of five amino acid residues that occur in P450 2C3 for the corresponding residues in P450 2C5 produced a protein P450 2C5/3LV that behaved as a soluble monomer. The protein crystallized readily and led to determination of the first structure of a mammalian microsomal cytochrome P450.
Eric F. Johnson