PDBsum entry 4zfa

Oxidoreductase

PDB id: 4zfa

Contents

Protein chain

460 a.a.

Ligands

HEM

1PE

EDO

Metals

_NI ×4

Waters ×57

PDB id:

4zfa

Name:

Oxidoreductase

Title:

Cytochrome p450 wild type from bm3 with bound peg

Structure:

Bifunctional p-450/NADPH-p450 reductase. Chain: a. Fragment: unp residues 1-461. Synonym: cytochrome p450(bm-3),cytochrome p450bm-3. Engineered: yes. Mutation: yes

Source:

Bacillus megaterium. Organism_taxid: 1404. Gene: cyp102a1, cyp102. Expressed in: escherichia coli. Expression_system_taxid: 511693.

Resolution:

2.77Å R-factor: 0.247 R-free: 0.302

Authors:

W.E.Rogers,T.Othman,D.K.Heidary,T.Huxford

Key ref:

I.Geronimo et al. (2016). Effect of Mutation and Substrate Binding on the Stability of Cytochrome P450BM3 Variants. Biochemistry, 55, 3594-3606. PubMed id: 27267136 DOI: 10.1021/acs.biochem.6b00183

Date:

21-Apr-15 Release date: 13-Jul-16

Protein chain

P14779  (CPXB_PRIM2) -  Bifunctional cytochrome P450/NADPH--P450 reductase from Priestia megaterium (strain ATCC 14581 / DSM 32 / CCUG 1817 / JCM 2506 / NBRC 15308 / NCIMB 9376 / NCTC 10342 / NRRL B-14308 / VKM B-512 / Ford 19)

Seq: 1049 a.a.

Struc: 460 a.a.

Enzyme reactions

• Enzyme class 2: E.C.1.14.14.1 - unspecific monooxygenase.
• Reaction: an organic molecule + reduced [NADPH--hemoprotein reductase] + O2 = an alcohol + oxidized [NADPH--hemoprotein reductase] + H2O + H⁺
• Cofactor: Heme-thiolate
• Enzyme class 3: E.C.1.6.2.4 - NADPH--hemoprotein reductase.
• Reaction: 2 oxidized [cytochrome P450] + NADPH = 2 reduced [cytochrome P450] + NADP⁺ + H⁺
• Cofactor: FAD; FMN

Note, where more than one E.C. class is given (as above), each may correspond to a different protein domain or, in the case of polyprotein precursors, to a different mature protein.

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

reference

DOI no: 10.1021/acs.biochem.6b00183

Biochemistry 55:3594-3606 (2016)

PubMed id: 27267136

Effect of Mutation and Substrate Binding on the Stability of Cytochrome P450BM3 Variants.

I.Geronimo, C.A.Denning, W.E.Rogers, T.Othman, T.Huxford, D.K.Heidary, E.C.Glazer, C.M.Payne.

ABSTRACT

Cytochrome P450BM3 is a heme-containing enzyme from Bacillus megaterium that exhibits high monooxygenase activity and has a self-sufficient electron transfer system in the full-length enzyme. Its potential synthetic applications drive protein engineering efforts to produce variants capable of oxidizing nonnative substrates such as pharmaceuticals and aromatic pollutants. However, promiscuous P450BM3 mutants often exhibit lower stability, thereby hindering their industrial application. This study demonstrated that the heme domain R47L/F87V/L188Q/E267V/F81I pentuple mutant (PM) is destabilized because of the disruption of hydrophobic contacts and salt bridge interactions. This was directly observed from crystal structures of PM in the presence and absence of ligands (palmitic acid and metyrapone). The instability of the tertiary structure and heme environment of substrate-free PM was confirmed by pulse proteolysis and circular dichroism, respectively. Binding of the inhibitor, metyrapone, significantly stabilized PM, but the presence of the native substrate, palmitic acid, had no effect. On the basis of high-temperature molecular dynamics simulations, the lid domain, β-sheet 1, and Cys ligand loop (a β-bulge segment connected to the heme) are the most labile regions and, thus, potential sites for stabilizing mutations. Possible approaches to stabilization include improvement of hydrophobic packing interactions in the lid domain and introduction of new salt bridges into β-sheet 1 and the heme region. An understanding of the molecular factors behind the loss of stability of P450BM3 variants therefore expedites site-directed mutagenesis studies aimed at developing thermostability.