Abstract
CYP4A11, the principal known human fatty acid omega-hydroxylase, has been expressed as a polyhistidine-tagged protein and purified to homogeneity. Based on an alignment with P450BM-3, the CYP4A11 L131F mutant has been constructed and similarly expressed. The two proteins are spectroscopically indistinguishable, but wild-type CYP4A11 primarily catalyzes omega-hydroxylation, and the L131F mutant only omega-1 hydroxylation, of lauric acid. The L131F mutant is highly uncoupled in that it slowly (omega-1)-hydroxylates lauric acid yet consumes NADPH at approximately the same rate as the wild-type enzyme. Wild-type CYP4A11 is inactivated by 1-aminobenzotriazole under turnover conditions but the L131F mutant is not. This observation, in conjunction with the binding affinities of substituted imidazoles for the two proteins, indicates that the L131F mutation decreases access of exogenous substrates to the heme site. Leu-131 thus plays a key role in controlling the regioselectivity of substrate hydroxylation and the extent of coupled versus uncoupled enzyme turnover. A further important finding is that the substituted imidazoles bind more weakly to CYP4A11 and its L131F mutant when these proteins are reduced by NADPH-cytochrome P450 reductase than by dithionite. This finding suggests that the ferric enzyme undergoes a conformational change that depends on both reduction of the iron and the presence of cytochrome P450 reductase and NADPH.
Highlights
CYP4A11, one of the three known members of the human CYP4 family [1,2,3,4], is the only established fatty acid -hydroxylase in human liver and kidney [5, 6]
The results (a) indicate that Leu-131, which corresponds to Phe-87 in P450BM-3, plays an important role in determining the hydroxylation regioselectivity, (b) provide evidence that CYP4A11 undergoes a P450 reductase-dependent conformational change on reduction of the heme iron atom, (c) unmask important differences in the susceptibility of CYP4A11 and CYP4A1 to inhibitors, and (d) strengthen the use of P450BM-3 as a model for the CYP4A family of proteins
Expression and Spectroscopic Characterization of CYP4A11 and L131F—The mutation to be made in CYP4A11 was chosen by alignment of the CYP4A11 sequence with that of P450BM-3, for which two independent crystal structures are available [18, 22]
Summary
CYP4A11, one of the three known members of the human CYP4 family [1,2,3,4], is the only established fatty acid -hydroxylase in human liver and kidney [5, 6]. The mechanism by which the -hydroxylation regiospecificity is enforced is not known, we recently reported that the CYP4A1 specificity is partially shifted toward (-1)-hydroxylation by a D323E mutation in the I-helix of the protein [15] Mutation of this residue was based on a sequence alignment of CYP4A1 with P450BM-3 (CYP102). The CYP4A11 Active Site giospecificity are obscured by the finding through NMR studies that the enzyme-substrate complex undergoes a major conformational change when the iron is reduced from the ferric to the ferrous state [24] This conformational change is reported to shift the position of the substrate relative to the iron by a distance of 6 Å. Despite the sequence and functional similarities between CYP4A11 and CYP4A1 and their mutual relationship to P450BM-3, important differences exist between the two CYP4A proteins
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
