Abstract
This report suggests an important physiological role of a CYP in the accumulation of uroporphyrin I arising from catalytic oxidative conversion of uroporphyrinogen I to uroporphyrin I in the periplasm of Escherichia coli cultured in the presence of 5-aminolevulinic acid. A structurally competent Streptomyces griseus CYP105D1 was expressed as an engineered, exportable form in aerobically grown E. coli. Its progressive induction in the presence of 5-aminolevulinic acid-supplemented medium was accompanied by an accumulation of a greater than 100-fold higher amount of uroporphyrin I in the periplasm relative to cells lacking CYP105D1. Expression of a cytoplasm-resident engineered CYP105D1 at a comparative level to the secreted form was far less effective in promoting porphyrin accumulation in the periplasm. Expression at a 10-fold molar excess over the exported CYP105D1 of another periplasmically exported hemoprotein, the globular core of cytochrome b5, did not substitute the role of the periplasmically localized CYP105D1 in promoting porphyrin production. This, therefore, eliminated the possibility that uroporphyrin accumulation is merely a result of increased hemoprotein synthesis. Moreover, in the strain that secreted CYP105D1, uroporphyrin production was considerably reduced by azole-based P450 inhibitors. Production of both holo-CYP105D1 and uroporphyrin was dependent upon 5-aminolevulinic acid, except that at higher concentrations this resulted in a decrease in uroporphyrin. This study suggests that the exported CYP105D1 oxidatively catalyzes periplasmic conversion of uroporphyrinogen I to uroporphyrin I in E. coli. The findings have significant implications in the ontogenesis of human uroporphyria-related diseases.
Highlights
Porphyrin metabolism in humans has been extensively investigated, in the occurrence of a group of clinically related diseases known as porphyrias, arising from genetic defects [1] of the enzymes in heme biosynthesis causing accumulation of various heme precursors [2, 3]
This report suggests an important physiological role of a CYP in the accumulation of uroporphyrin I arising from catalytic oxidative conversion of uroporphyrinogen I to uroporphyrin I in the periplasm of Escherichia coli cultured in the presence of 5-aminolevulinic acid
The soluble CYP105D1 of Streptomyces griseus has been successfully expressed in Escherichia coli as an exportable form that was efficiently localized to the periplasm [15]
Summary
All chemicals were purchased from Sigma. Clotrimazole, triadimefon, and miconazole were kindly donated by Dr E. The CYP105D1 enzyme was purified from the periplasmic extract of E. coli CYP105D1 (1 liter of culture) cultivated for 24 h at 28 °C with 1 mM ALA supplementation. Following 2-fold dilution of the periplasmic extract with 10 mM Tris acetate (pH 8) buffer (TA) containing 1 mM EDTA, the sample was applied twice through a TA-pre-equilibrated column of DEAE CL-6B-Sepharose (5-ml bed volume). Isolation and Estimation of URO—Periplasmic and cytoplasmic extracts were derived from E. coli cultured in 100 ml of MOPS medium supplemented with ALA and 75 g/ml ampicillin. For the analysis and estimation of URO by HPLC [22], the periplasmic fraction (5-ml volume recovered from 100-ml culture) was first deproteinated with 0.2 M HCl as described above. Following 5-fold dilution with distilled water, the solution was applied onto a DEAE-Sepharose CL-6B column (1-ml bed volume). To test the ability of the E. coli TB1 strain to utilize exogenous heme, the stock heme solution was applied in the MOPS medium at 5-fold increasing concentrations ranging from 1 to 50 M
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