PURIFICATION AND CHARACTERIZATION OF RECOMBINANT CYTOCHROME P450(TYR) EXPRESSED AT HIGH-LEVELS IN ESCHERICHIA-COLI

The multifunctional tyrosine N-hydroxylase, cytochrome P450(TYR) (CYP79), from Sorghum bicolor catalyzing the conversion of tyrosine to p-hydroxyphenylacetaldoxime 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 P450(TYR) and by exchanging the first eight codons with the first eight codons of bovine P45017 alpha. The highest yielding construct provided 200-500 nmol P450(TYR)/liter cell culture, The recombinant P450(TYR) 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 K-m and turnover rate for isolated recombinant P450(TYR) 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 P450(TYR) 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 P450(TYR), 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 P450(TYR).