Functional characterization of cytochromes P450 2B from the desert woodrat Neotoma lepida

Abstract

Mammalian detoxification processes have been the focus of intense research, but little is known about how wild herbivores process plant secondary compounds, many of which have medicinal value or are drugs. cDNA sequences that code for three enzymes of the cytochrome P450 (CYP) 2B subfamily, here termed 2B35, 2B36, and 2B37 have been recently identified from a wild rodent, the desert woodrat (Malenke et al., 2012). Two variant clones of each enzyme were engineered to increase protein solubility and to facilitate purification, as reported for CYP2B enzymes from multiple species. When expressed in Escherichia coli each of the woodrat proteins gave the characteristic maximum at 450nm in a reduced carbon monoxide difference spectrum but generally expressed at lower levels than rat CYP2B1. Two enzymes, 2B36 and 2B37, showed dealkylation activity with the model substrates 7-ethoxy-4-(trifluoromethyl)coumarin and 7-benzyloxyresorufin, whereas 2B35 was inactive. Binding of the monoterpene (+)-α-pinene produced a Type I shift in the absorbance spectrum of each enzyme. Mutation of 2B37 at residues 114, 262, or 480, key residues governing ligand interactions with other CYP2B enzymes, did not significantly change expression levels or produce the expected functional changes. In summary, two catalytic and one ligand-binding assay are sufficient to distinguish among CYP2B35, 2B36, and 2B37. Differences in functional profiles between 2B36 and 2B37 are partially explained by changes in substrate recognition site residue 114, but not 480. The results advance our understanding of the mechanisms of detoxification in wild mammalian herbivores and highlight the complexity of this system.

Keywords: 2-mercaptoethanol; 2BXdH; 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; 7-BR; 7-EFC; 7-benzyloxyresorufin; 7-ethoxy-4-(trifluoromethyl)coumarin; BME; Biotransformation; CHAPS; CYP; Cytochrome P450; DTT; DXMS; Detoxification; EDTA; HS(P450); ITC; Juniper; PMSF; PSC; Woodrats; an N-terminally truncated and modified 2B enzyme with a C-terminal tetra-His tag to facilitate purification; cytochrome P450-dependent monooxygenases; dithiotheitol; ethylenediaminetetraacetic acid; high spin P450; hydrogen–deuterium exchange coupled to mass spectrometry; isothermal titration calorimetry; phenylmethanesulfonyl fluoride; plant secondary compound or plant specialized compound.