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J. Biol. Chem., Vol. 280, Issue 32, 29217-29223, August 12, 2005

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Key Role of Conserved Histidines in Recombinant Mouse -Carotene 15,15'-Monooxygenase-1 Activity^*

Eugenia Poliakov, Susan Gentleman, Francis X. Cunningham, Jr., Nancy J. Miller-Ihli¶, and T. Michael Redmond||

From the Laboratory of Retinal Cell and Molecular Biology, NEI, National Institutes of Health, Bethesda, Maryland 20892-0706, the Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland 20742, and the ^¶Food Composition Laboratory, Agricultural Research Service, United States Department of Agriculture, Beltsville, Maryland 20705

Alignment of sequences of vertebrate -carotene 15,15'-monooxygenase-1 (BCMO1) and related oxygenases revealed four perfectly conserved histidines and five acidic residues (His¹⁷², His²³⁷, His³⁰⁸, His⁵¹⁴, Asp⁵², Glu¹⁴⁰, Glu³¹⁴, Glu⁴⁰⁵, and Glu⁴⁵⁷ in mouse BCMO1). Because BCMO1 activity is iron-dependent, we propose that these residues participate in iron coordination and therefore are essential for catalytic activity. To test this hypothesis, we produced mutant forms of mouse BCMO1 by replacing the conserved histidines and acidic residues as well as four histidines and one glutamate non-conserved in the overall family with alanines by site-directed mutagenesis. Our in vitro and in vivo data showed that mutation of any of the four conserved histidines and Glu⁴⁰⁵ caused total loss of activity. However, mutations of non-conserved histidines or any of the other conserved acidic residues produced impaired although enzymatically active proteins, with a decrease in activity mostly due to changes in V_max. The iron bound to protein was determined by inductively coupled plasma atomic emission spectrometry. Bound iron was much lower in preparations of inactive mutants than in the wild-type protein. Therefore, the conserved histidines and Glu⁴⁰⁵ are absolutely required for the catalytic mechanism of BCMO1. Because the mutant proteins are impaired in iron binding, these residues are concluded to coordinate iron required for catalytic activity. These data are discussed in the context of the predicted structure for the related eubacterial apocarotenal oxygenase.

Received for publication, January 12, 2005 , and in revised form, June 2, 2005.

^* 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: NEI, LRCMB, NIH, Bldg. 7, Rm. 303, 7 Memorial Dr., MSC 0706, Bethesda, MD 20892-0706. Tel.: 301-496-0439; Fax: 301-402-0750; E-mail: Redmond{at}helix.nih.gov.

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