Functional evaluation of cytochrome P450 2D6 with Gly42Arg substitution expressed in Saccharomyces cerevisiae

Abstract

A single amino acid-substituted mutant protein, CYP2D6 (G42R) was expressed in Saccharomyces cerevisiae and its enzymatic properties were compared with those of other single (P34S, R296C and S486T) and double amino acid-substituted mutant proteins (P34S/S486T and R296C/S486T) expressed in yeast cells, all of which were known to occur in the CYP2D6 gene as single nucleotide polymorphisms. The protein levels of G42R, P34S and P34S/S486T in microsomal fractions and their oxidation capacities towards debrisoquine as a prototypic substrate and bunitrolol as a chiral substrate were different from those of wild-type CYP2D6, while the R296C, S486T and R296C/S486T behaved similarly to the wild-type in these indices. The CYP contents both in yeast microsomal and in whole cell fractions indicated that some part of G42R protein was localized in the endoplasmic reticulum membrane fraction, whereas most of G42R protein was in some subcellular fractions other than endoplasmic reticulum. In kinetic analysis, the G42R substitution increased apparent Km and decreased Vmax for debrisoquine 4-hydroxylation, while it increased both Km and Vmax for bunitrolol 4-hydroxylation. The P34S substitution did not drastically change Km but decreased Vmax for debrisoquine 4-hydroxylation, whereas Km was increased and Vmax unchanged or decreased for bunitrolol 4-hydroxylation by P34S substitution. These results suggest that the G42R substitution causes a change in the CYP2D6 conformation, which may be different from the change produced by the P34S substitution.

Genetic polymorphism, linkage disequilibrium, haplotype structure and novel allele analysis of CYP2C19 and CYP2D6 in Han Chinese

“…Previous findings indicated that substitution located in the N-terminal region-such as G42R 49,50 and P34S…”

“…50 Similarly, lower enzyme activity was observed in G42E substitution with lower microsomal contents and higher K m value for oxidation of debrisoquine than the wild type, due to lower efficiency of the protein in its anchoring to endoplasmic reticulum membranes. 53 Therefore, CYP2D6*71 could be a putative PM allele.…”

Changes in the Enzymatic Properties of CYP2D6 by the Substitution of Phenylalanine at Position 120 by Alanine

“…7) Briefly, the mutation-bearing plasmid was digested with HindIII, and the resultant fragment was inserted into HindIII-digested dephosphorylated pGYRI and transformed into Escherichia coli (E. coli) HB101. The yeast expression vector pGYRI has a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoter and includes the yeast NADPH-CYP reductase gene.…”

“…[4][5][6] In our previous project, CYP2D2 (P450BTL) was purified employing bunitrolol (BTL) 4-hydroxylase as the index of CYP2D2 functions. 4) BTL is a β-adrenoceptor blocking agent, and rat CYP2D2, 4) as well as human CYP2D6, 7) catalyzes BTL 4-hydroxylation with relatively low Km values. Our interest has also been directed to the mechanism(s) responsible for the stereoselectivity in the oxidation of various chiral substrates by CYP2D enzymes.…”

“…8) For example, rat CYP2D2 shows substrate enantioselectivity of [(+)-BTL < (-)-BTL], 4) whereas human CYP2D6 exhibits the reverse selectivity of [(+)-BTL > (-)-BTL]. 7) As part of our overall studies, the present study was conducted to determine what amino acid residues in the active site of human CYP2D6 are involved in the enantioselective oxidation of BTL enantiomers by the enzyme. In the search of the active site, it seems likely that the phenyl ring of phe- nylalanine at position 120 (Phe-120) may affect the positioning of the aromatic ring of the substrate in the active site of CYP2D6, giving the idea that the substitution of Phe-120 by another amino acid residue may change the activity or substrate enantioselectivity in BTL 4-hydroxylation by CYP2D6.…”