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

Geronimo, Inacrist; Denning, Catherine A.; Rogers, W. Eric; Othman, Thaer; Huxford, Tom; Heidary, David K.; Glazer, Edith C.; Payne, Christina M.

doi:10.1021/acs.biochem.6b00183

Title: Effect of Mutation and Substrate Binding on the Stability of Cytochrome P450_BM3 Variants

Journal Article · Mon Jun 06 00:00:00 EDT 2016 · Biochemistry

DOI:https://doi.org/10.1021/acs.biochem.6b00183· OSTI ID:1260843

Geronimo, Inacrist ^[1]; Denning, Catherine A. ^[1]; Rogers, W. Eric ^[2]; Othman, Thaer ^[2]; Huxford, Tom ^[2]; Heidary, David K. ^[1]; Glazer, Edith C. ^[1]; Payne, Christina M. ^[1]

Univ. of Kentucky, Lexington, KY (United States)
San Diego State Univ., CA (United States)

Cytochrome P450_BM3 is a heme-containing enzyme from Bacillus megaterium that displays 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, 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 P450_BM3 variants therefore expedites site-directed mutagenesis studies aimed at developing thermostability.

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Research Organization:: Argonne National Laboratory (ANL), Argonne, IL (United States). Advanced Photon Source (APS)

Sponsoring Organization:: National Institute of General Medical Sciences (NIGMS); National Institutes of Health (NIH); Office of Research Infrastructure Programs (ORIP); USDOE Office of Science (SC); National Science Foundation (NSF); National Institute on Drug Abuse (NIDA)

Grant/Contract Number:: P41 GM103403; S10 RR029205; AC02-06CH11357; ACI-1053575; 1355438; California Metabolic Research Foundation

OSTI ID:: 1260843

Journal Information:: Biochemistry, Vol. 55, Issue 25; ISSN 0006-2960

Publisher:: American Chemical Society (ACS)Copyright Statement

Country of Publication:: United States

Language:: ENGLISH

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Cited by: 12 works

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

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Title: Effect of Mutation and Substrate Binding on the Stability of Cytochrome P450_BM3 Variants