PDBsum entry 4l4c

Oxidoreductase

PDB id: 4l4c

Contents

Protein chains

405 a.a.

Ligands

HEM ×2

CAM ×2

Metals

__K ×3

Waters ×544

PDB id:

4l4c

Name:

Oxidoreductase

Title:

Structure of l358p/k178g mutant of p450cam bound to camphor

Structure:

Camphor 5-monooxygenase. Chain: a, b. Synonym: cytochrome p450-cam, cytochrome p450cam. Engineered: yes. Mutation: yes

Source:

Pseudomonas putida. Organism_taxid: 303. Gene: camc, cyp101. Expressed in: escherichia coli. Expression_system_taxid: 562

Resolution:

2.20Å R-factor: 0.174 R-free: 0.243

Authors:

D.Batabyal,H.Li,T.L.Poulos

Key ref:

D.Batabyal et al. (2013). Synergistic effects of mutations in cytochrome P450cam designed to mimic CYP101D1. Biochemistry, 52, 5396-5402. PubMed id: 23865948 DOI: 10.1021/bi400676d

Date:

07-Jun-13 Release date: 31-Jul-13

Protein chains

P00183  (CPXA_PSEPU) -  Camphor 5-monooxygenase from Pseudomonas putida

Seq: 415 a.a.

Struc: 405 a.a.*

* PDB and UniProt seqs differ at 3 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.1.14.15.1 - camphor 5-monooxygenase.
• Reaction: 2 reduced [2Fe-2S]-[putidaredoxin] + (1R,4R)-camphor + O2 + 2 H⁺ = (1R,4R,5R)-5-hydroxycamphor + 2 oxidized [2Fe-2S]-[putidaredoxin] + H2O
• Cofactor: Heme-thiolate

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

Key reference

DOI no: 10.1021/bi400676d

Biochemistry 52:5396-5402 (2013)

PubMed id: 23865948

Synergistic effects of mutations in cytochrome P450cam designed to mimic CYP101D1.

D.Batabyal, H.Li, T.L.Poulos.

ABSTRACT

A close orthologue to cytochrome P450cam (CYP101A1) that catalyzes the same hydroxylation of camphor to 5-exo-hydroxycamphor is CYP101D1. There are potentially important differences in and around the active site that could contribute to subtle functional differences. Adjacent to the heme iron ligand, Cys357, is Leu358 in P450cam, whereas this residue is Ala in CYP101D1. Leu358 plays a role in binding of the P450cam redox partner, putidaredoxin (Pdx). On the opposite side of the heme, about 15-20 Å away, Asp251 in P450cam plays a critical role in a proton relay network required for O2 activation but forms strong ion pairs with Arg186 and Lys178. In CYP101D1 Gly replaces Lys178. Thus, the local electrostatic environment and ion pairing are substantially different in CYP101D1. These sites have been systematically mutated in P450cam to the corresponding residues in CYP101D1 and the mutants analyzed by crystallography, kinetics, and UV-vis spectroscopy. Individually, the mutants have little effect on activity or structure, but in combination there is a major drop in enzyme activity. This loss in activity is due to the mutants being locked in the low-spin state, which prevents electron transfer from the P450cam redox partner, Pdx. These studies illustrate the strong synergistic effects on well-separated parts of the structure in controlling the equilibrium between the open (low-spin) and closed (high-spin) conformational states.