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Vol. 290, Issue 3, 1141-1147, September 1999
Department of Pharmacology and Toxicology, University of Texas
Medical Branch, Galveston, Texas (T.L.D., F.R., J.R.H.); and Department
of Drug Metabolism and Pharmacokinetics, Rhône-Poulenc Rorer,
Collegeville, Pennsylvania (K.M.S., J.C.S.)
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.
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