Structure-function analysis of human cytochrome P-450 2B6 using a novel substrate, site-directed mutagenesis, and molecular modeling.
The structural basis for functional differences between human cytochrome P-450 2B6 and rat 2B1 was investigated. An amino acid sequence alignment predicted the location of 2B6 substrate recognition site (SRS) residues. Ten residues within these SRSs unique to 2B6 compared with 2B1, 2B4, and 2B11 were chosen for mutagenesis. Two additional sites that differ between 2B6 and 2B1 and are known to have a role in 2B1 substrate specificity were also mutated. The 2B6 mutants were expressed in Spodoptera frugiperda cells and characterized using the 2B6-specific substrate RP 73401 [3-cyclopentyloxy-N-(3,5-dichloro-4-pyridyl)-4-methoxybenzamide], the 2B1-selective substrate androstenedione, and the common substrate 7-ethoxy-4-trifluoromethylcoumarin. Mutants F107I and L363V exhibited decreased RP 73401 hydroxylation but retained most of the wild-type level of 2B6 7-ethoxy-4-trifluoromethylcoumarin O-deethylase activity. In addition, SRS exchanges were studied in which the amino acid sequence of 2B6 SRSs was converted to the sequence of 2B1. Each of these constructs, having two to seven substitutions, expressed at levels similar to 2B6 but did not acquire significant androstenedione hydroxylase activity. Docking of RP 73401 into the active site of a 2B6 homology model suggested a direct interaction with residue L363 but not with F107. Findings from this study suggest that 1) residues F107 and L363 are necessary for 2B6 RP 73401 hydroxylase activity, 2) 2B6 is able to tolerate multiple SRS substitutions without compromising protein expression levels or protein stability, and 3) conferring androstenedione hydroxylase function to cytochrome P-450 2B6 is more complex than altering a single SRS.
Domanski
T L
TL
Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, Texas, USA. tadomans@utmb.edu
Schultz
K M
KM
Roussel
F
F
Stevens
J C
JC
Halpert
J R
JR
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D016367:Baculoviridae / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D065702:Cytochrome P-450 CYP2B6; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004789:Enzyme Activation / Q000235:genetics; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010089:Oxidoreductases, N-Demethylating / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D051381:Rats; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012984:Software; D018411:Spodoptera / Q000235:genetics / Q000821:virology; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D003577:Cytochrome P-450 Enzyme System; D001189:Aryl Hydrocarbon Hydroxylases; C585599:CYP2B6 protein, human; D065702:Cytochrome P-450 CYP2B6; D010089:Oxidoreductases, N-Demethylating