Purification and characterization of recombinant cytochrome P450TYR expressed at high levels in Escherichia coli.
The multifunctional tyrosine N-hydroxylase, cytochrome P450TYR (CYP79), from Sorghum bicolor catalyzing the conversion of tyrosine to p-hydroxyphenyl-acetaldoxime in the biosynthesis of the cyanogenic glucoside dhurrin, has been expressed in Escherichia coli using the isopropyl-beta-D-thiogalactopyranoside-inducible vector pSP19g10L, containing the cDNA encoding CYP79. The expression construct was optimized by reducing the length of the N-terminal hydrophobic core of the signal sequence of cytochrome P450TYR and by exchanging the first eight codons with the first eight codons of bovine P45017 alpha. The highest yielding construct provided 200-500 nmol P450TYR/liter cell culture. The recombinant P450TYR was gently and efficiently extracted from E. coli spheroblasts by temperature-induced phase partitioning of Triton X-114 in the presence of 30% glycerol and isolated by DEAE and reactive red chromatography. In reconstitution experiments using saturating amounts of sorghum NADPH-cytochrome P450 reductase, the Km and turnover rate for isolated recombinant P450TYR was 0.22 +/- 0.06 mM and 49.2 +/- 3.8 min-1, respectively, whereas a turnover rate as high as 350 min-1, was obtained using E. coli membranes. Addition of 3 mM glutathione stimulated the activity of reconstituted P450TYR and of sorghum microsomes although the effect was highly variable. Phenylalanine, the precursor of several cyanogenic glucosides, gave a type I binding spectrum, but was not metabolized by P450TYR, demonstrating the high substrate specificity of this P450. Administration of radioactively labeled p-hydroxyphenylacetaldoxime to E. coli cells, showed E. coli metabolized p-hydroxyphenylacetaldoxime independent of the expression of P450TYR.
Halkier
B A
BA
Department of Plant Biology, Royal Veterinary and Agricultural University, Copenhagen, Denmark.
Nielsen
H L
HL
Koch
B
B
Møller
B L
BL
D000595:Amino Acid Sequence; D001483:Base Sequence; D002451:Cell Compartmentation; D002458:Cell Fractionation; D003577:Cytochrome P-450 Enzyme System / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005978:Glutathione / Q000494:pharmacology; D008861:Microsomes / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D006109:Poaceae / Q000201:enzymology* / Q000235:genetics*; D011994:Recombinant Proteins / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry
D010940:Plant Proteins; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C089817:cytochrome P450TYR; D005978:Glutathione