Effect of Mutation and Substrate Binding on the Stability of Cytochrome P450(BM3) Variants

Abstract

Cytochrome P450(BM3) 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 P450(BM3) 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, beta-sheet 1, and Cys ligand loop (a beta-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 beta-sheet 1 and the heme region. An understanding of the molecular factors behind the loss of stability of P450(BM3) variants therefore expedites site-directed mutagenesis studies aimed at developing thermostability.

Published in

Biochemistry
2016, Volume: 55, number: 25, pages: 3594-3606
Publisher: AMER CHEMICAL SOC

UKÄ Subject classification

Theoretical Chemistry
Biochemistry and Molecular Biology


Publication identifier

DOI: https://doi.org/10.1021/acs.biochem.6b00183

Permanent link to this page (URI)

https://res.slu.se/id/publ/89953