PDBsum entry 4lht

Oxidoreductase

PDB id: 4lht

Contents

Protein chains

396 a.a.

Ligands

HEM ×2

CNL ×2

PEG

PGE

SO4

PG4

Metals

_CL

Waters ×423

PDB id:

4lht

Name:

Oxidoreductase

Title:

Crystal structure of p450cin y81f mutant, crystallized in 3 mm 1,8- cineole

Structure:

P450cin. Chain: a, b. Fragment: unp residues 8-404. Engineered: yes. Mutation: yes

Source:

Citrobacter braakii. Organism_taxid: 57706. Gene: cin a, cina. Expressed in: escherichia coli. Expression_system_taxid: 668369.

Resolution:

2.14Å R-factor: 0.212 R-free: 0.263

Authors:

Y.Madrona,T.L.Poulos

Key ref:

Y.Madrona et al. (2013). P450cin active site water: implications for substrate binding and solvent accessibility. Biochemistry, 52, 5039-5050. PubMed id: 23829586 DOI: 10.1021/bi4006946

Date:

01-Jul-13 Release date: 24-Jul-13

Protein chains

Q8VQF6  (CINA_CITBR) -  1,8-cineole 2-endo-monooxygenase from Citrobacter braakii

Seq: 404 a.a.

Struc: 396 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class: E.C.1.14.14.133 - 1,8-cineole 2-endo-monooxygenase.
• Reaction: 1,8-cineole + reduced [flavodoxin] + O2 = 2-endo-hydroxy-1,8-cineole + oxidized [flavodoxin] + H2O + H⁺

1,8-cineole (Bound ligand (Het Group name = CNL) corresponds exactly) + reduced [flavodoxin] + O2 = 2-endo-hydroxy-1,8-cineole + oxidized [flavodoxin] + H2O + H(+)

• Cofactor: Heme-thiolate

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

Added reference

DOI no: 10.1021/bi4006946

Biochemistry 52:5039-5050 (2013)

PubMed id: 23829586

P450cin active site water: implications for substrate binding and solvent accessibility.

Y.Madrona, S.A.Hollingsworth, B.Khan, T.L.Poulos.

ABSTRACT

In P450cin, Tyr81, Asp241, Asn242, two water molecules, and the substrate participate in a complex H-bonded network. The role of this H-bonded network in substrate binding and catalysis has been probed by crystallography, spectroscopy, kinetics, isothermal titration calorimetry (ITC), and molecular dynamics. For the Y81F mutant, the substrate binds about 20-fold more weakly and Vmax decreases by about 30% in comparison to WT. The enhanced susceptibility of the heme to H2O2-mediated destruction in Y81F suggests that this mutant favors the open, low-spin conformational state. Asn242 H-bonds directly with the substrate, and replacing this residue with Ala results in water taking the place of the missing Asn side chain. This mutant exhibits a 70% decrease in activity. Crystal structures and molecular dynamics simulations of substrate-bound complexes show that the solvent has more ready access to the active site, especially for the N242A mutant. This accounts for about a 64% uncoupling of electron transfer from substrate hydroxylation. These data indicate the importance of the interconnected water network on substrate binding and on the open/closed conformational equilibrium, which are both critically important for maintaining high-coupling efficiency.