Mutagenesis study of Asp-290 in cytochrome P450 2B11 using a fusion protein with rat NADPH-cytochrome P450 reductase.
Asp-290 of the phenobarbital-inducible dog liver cytochrome P450 (P450) 2B11 was mutated to nine other amino acid residues by site-directed mutagenesis, and the functional significance of the unique negative charge in P450 2B11 at that position was studied. To facilitate the analysis of mutated P450 2B11 enzymes heterologously expressed in Escherichia coli, an enzymatically active fusion enzyme was genetically engineered between the cDNAs for P450 2B11 and rat liver NADPH-cytochrome P450 reductase using a Ser-Thr linker as previously described (Fisher et al., 1992, Proc. Natl. Acad. Sci. USA 89, 10817-10821). Sonicated whole-cell lysates of E. coli cells expressing the wild-type fusion protein were able to catalyze the 16-hydroxylation of androstenedione (AD) in the absence of added reductase, and exhibited activities and androstenedione metabolite profiles very similar to those of purified and reconstituted enzyme preparations. The substitution of Ala, Glu, Gly, Met, Asn, Arg, Ser, Thr, or Val for Asp-290 of P450 2B11 resulted in decreased AD hydroxylase activities as assessed using solubilized membranes. Replacement of Asp-290 with Glu yielded the highest activity (55% of wild type), while substituting the positively charged amino acid Arg created an enzyme with the lowest activity (< 1% of wild-type activity). Regioselectivity of AD hydroxylation was not affected although the stereoselectivity of hydroxylation at the 16 carbon position was altered in some cases. The use of the fused enzyme to study the effects of site-directed mutagenesis has resulted in the demonstration of the importance of size and charge at position 290 for enzymatic activity of P450 2B11.
Harlow
G R
GR
Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson 85721, USA. Harlow@tonic.pharm.arizona.edu
Halpert
J R
JR
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics*; D001216:Asparagine / Q000235:genetics; D001483:Base Sequence; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D000072467:Cytochrome P450 Family 2; D004285:Dogs; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009251:NADPH-Ferrihemoprotein Reductase; D010088:Oxidoreductases / Q000235:genetics*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics; D013250:Steroid Hydroxylases
D011993:Recombinant Fusion Proteins; D001216:Asparagine; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; D000072467:Cytochrome P450 Family 2; C090278:cytochrome P-450 CYP2B11 (dog); D009251:NADPH-Ferrihemoprotein Reductase