4ZFB

Cytochrome P450 pentamutant from BM3 bound to Palmitic Acid

Summary for 4ZFB

• Entry DOI: 10.2210/pdb4zfb/pdb
• Related: 4ZF6 4ZF8 4ZF9 4ZFA 4ZFD 4ZFE
• Descriptor: Bifunctional P-450/NADPH-P450 reductase, PROTOPORPHYRIN IX CONTAINING FE, PALMITIC ACID, ... (6 entities in total)
• Functional Keywords: cytochrome p450, heme oxidase domain, oxidoreductase, bacillus megaterium
• Biological source: Bacillus megaterium
• Cellular location: Cytoplasm : P14779
• Total number of polymer chains: 1
• Total formula weight: 54670.72

Authors

Rogers, W.E.,Othman, T.,Heidary, D.K.,Huxford, T. (deposition date: 2015-04-21, release date: 2016-07-13, Last modification date: 2023-09-27)

Primary citation

Geronimo, I.,Denning, C.A.,Rogers, W.E.,Othman, T.,Huxford, T.,Heidary, D.K.,Glazer, E.C.,Payne, C.M.
Effect of Mutation and Substrate Binding on the Stability of Cytochrome P450BM3 Variants.
Biochemistry, 55:3594-3606, 2016

PubMed 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.

PubMed: 27267136
DOI: 10.1021/acs.biochem.6b00183

Experimental method

X-RAY DIFFRACTION (2.84 Å)