The role of cytochrome P450 BM3 phenylalanine-87 and threonine-268 in binding organic hydroperoxides

• Published in: Biochimica et biophysica acta Vol. 1860; no. 4; pp. 669 - 677
• Main Authors: Roberts, Arthur G., Katayama, Jonathan, Kaspera, Rüdiger, Ledwitch, Kaitlyn V., Le Trong, Isolde, Stenkamp, Ronald E., Thompson, John A., Totah, Rheem A.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.04.2016
• Subjects: active sites; Bacillus megaterium; Bacillus megaterium - enzymology; Bacillus megaterium - genetics; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Binding Sites; chemical reactions; Computer modeling; cytochrome P-450; Cytochrome P-450 Enzyme System - chemistry; Cytochrome P-450 Enzyme System - genetics; Cytochrome P450 102A1; Cytochrome P450 BM3; heme; hydroperoxides; mammals; metabolism; molecular models; mutants; mutation; NADPH-Ferrihemoprotein Reductase - chemistry; NADPH-Ferrihemoprotein Reductase - genetics; Organic hydroperoxides; Phenylalanine - chemistry; Phenylalanine - genetics; Protein Binding; Quinones - chemistry; Structure-Activity Relationship; Substrate specificity/selectivity; Threonine - chemistry; Threonine - genetics; X-radiation; X-ray crystallography; X-ray diffraction; X-ray crystallography; Cytochrome P450 BM3; Cytochrome P450 102A1; Organic hydroperoxides; Substrate specificity/selectivity; Computer modeling
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/j.bbagen.2015.12.014

Cytochrome P450 (P450) BM3, from Bacillus megaterium, catalyzes a wide range of chemical reactions and is routinely used as a model system to study mammalian P450 reactions and structure. The metabolism of 2,6-di-tert-butyl-4-hydroperoxy-4-methyl-2,5-cyclohexadienone (BHTOOH) and 2-tert-butyl-4-hydroperoxy-4-methyl-2,5-cyclohexadien-1-one (BMPOOH) was examined with P450 BM3 and with the conserved T268 and F87 residues mutated to investigate their effects on organic hydroperoxide metabolism. To determine the effects of the mutations on the active site volume and architecture, the X-ray crystal structure of the F87A/T268A P450 BM3 heme domain (BMP) was determined and compared to previous structures. To investigate the interactions of the substrates with the F87 and T268 residues, BHTOOH and BMPOOH were docked into the BMP X-ray crystal structures. Lower metabolism of BHTOOH and BMPOOH was observed in the WT P450 BM3 and the T268A P450 BM3 mutant than in the F87A and F87A/T268A P450 BM3 mutants. Large differences were found in the F–G loop regions and active site cavity volumes for the F87A mutated structures. Analysis of the metabolism, X-ray crystal structures, and molecular docking simulations suggests that P450 BM3 activity toward BHTOOH and BMPOOH is mediated through substrate recognition by T268 and F87, and the active site cavity volume. Based on this information, a simplified representation is presented with the relative orientation of organic hydroperoxides in the P450 BM3 active site. The metabolism results and structural analysis of this model P450 allowed us to rationalize the structural factors that influence organic hydroperoxide metabolism. [Display omitted] •P450 BM3 is a model P450 system to study organic hydroperoxide metabolism.•P450 BM3 with the F87A mutation had higher activity toward organic hydroperoxides.•F87 and T268 are responsible for recognition of organic hydroperoxides.•F87, T268, and active site volume influence hydroperoxide binding and metabolism.