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J. Biol. Chem., Vol. 278, Issue 46, 45555-45562, November 14, 2003

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Export of a Heterologous Cytochrome P450 (CYP105D1) in Escherichia coli Is Associated with Periplasmic Accumulation of Uroporphyrin^*

M. Kalim Akhtar, Naheed N. Kaderbhai, David J. Hopper, Steven L. Kelly, and Mustak A. Kaderbhai

From the Institute of Biological Sciences, Cledwyn Building, University of Wales, Aberystwyth, Ceredigion, Wales SY23 3DD, United Kingdom

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.

Received for publication, December 12, 2002 , and in revised form, August 15, 2003.

^* This work was supported in part by the Biotechnology and Biological Science Research Council. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 44-1970-622294; Fax: 44-1970-622294; E-mail: mak{at}aber.ac.uk.

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This article has been cited by other articles:

				M. A. Kaderbhai, H. M. Davey, and N. N. Kaderbhai A directed evolution strategy for optimized export of recombinant proteins reveals critical determinants for preprotein discharge Protein Sci., September 1, 2004; 13(9): 2458 - 2469. [Abstract] [Full Text] [PDF]