Effect of Mutation and Substrate Binding on the Stability of Cytochrome P450BM3 Variants.
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.
Department of Chemical and Materials Engineering, University of Kentucky , Lexington, Kentucky 40506-0046, United States.
Inacrist
I
Geronimo
Department of Chemistry, University of Kentucky , Lexington, Kentucky 40506-0055, United States.
Catherine A
CA
Denning
Structural Biochemistry Laboratory, Department of Chemistry & Biochemistry, San Diego State University , San Diego, California 92182-1030, United States.
W Eric
WE
Rogers
Structural Biochemistry Laboratory, Department of Chemistry & Biochemistry, San Diego State University , San Diego, California 92182-1030, United States.
Thaer
T
Othman
Structural Biochemistry Laboratory, Department of Chemistry & Biochemistry, San Diego State University , San Diego, California 92182-1030, United States.
Tom
T
Huxford
Department of Chemistry, University of Kentucky , Lexington, Kentucky 40506-0055, United States.
David K
DK
Heidary
Department of Chemistry, University of Kentucky , Lexington, Kentucky 40506-0055, United States.
Edith C
EC
Glazer
Department of Chemical and Materials Engineering, University of Kentucky , Lexington, Kentucky 40506-0046, United States.
Christina M
CM
Payne
D001410:Bacillus megaterium / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D002942:Circular Dichroism; D003460:Crystallization; D018360:Crystallography, X-Ray; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004579:Electron Transport; D004791:Enzyme Inhibitors / Q000378:metabolism; D006900:Hydroxylation; D007700:Kinetics; D008797:Metyrapone / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009154:Mutation / Q000235:genetics*; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010084:Oxidation-Reduction; D019308:Palmitic Acid / Q000378:metabolism*; D011487:Protein Conformation
D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D050505:Mutant Proteins; D019308:Palmitic Acid; D003577:Cytochrome P-450 Enzyme System; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases; D008797:Metyrapone