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J. Biol. Chem., Vol. 279, Issue 37, 38960-38968, September 10, 2004

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Crystal Structure of the Oxygen-dependant Coproporphyrinogen Oxidase (Hem13p) of Saccharomyces cerevisiae^*

John D. Phillips¶, Frank G. Whitby||, Christy A. Warby, Pierre Labbe**, Cheng Yang, James W. Pflugrath, Joseph D. Ferrara, Howard Robinson, James P. Kushner, and Christopher P. Hill||¶¶

From the Departments of Medicine and ^||Biochemistry, University of Utah School of Medicine, Salt Lake City, Utah 84132, the ^**Laboratoire de Biochimie des Porphyrines, Institut Jacques Monod CNRS, Université Paris 7, 2 place Jussieu, 75251 Paris Cedex 5, France, Rigaku/Molecular Structure Corporation, The Woodlands, Texas 77381, and the Biology Department, Brookhaven National Laboratory, Upton, New York 11973-5000

Coproporphyrinogen oxidase (CPO) is an essential enzyme that catalyzes the sixth step of the heme biosynthetic pathway. Unusually for heme biosynthetic enzymes, CPO exists in two evolutionarily and mechanistically distinct families, with eukaryotes and some prokaryotes employing members of the highly conserved oxygen-dependent CPO family. Here, we report the crystal structure of the oxygen-dependent CPO from Saccharomyces cerevisiae (Hem13p), which was determined by optimized sulfur anomalous scattering and refined to a resolution of 2.0 Å. The protein adopts a novel structure that is quite different from predicted models and features a central flat seven-stranded anti-parallel sheet that is flanked by helices. The dimeric assembly, which is seen in different crystal forms, is formed by packing of helices and a short isolated strand that forms a -ladder with its counterpart in the partner subunit. The deep active-site cleft is lined by conserved residues and has been captured in open and closed conformations in two different crystal forms. A substratesized cavity is completely buried in the closed conformation by the 8-Å movement of a helix that forms a lid over the active site. The structure therefore suggests residues that likely play critical roles in catalysis and explains the deleterious effect of many of the mutations associated with the disease hereditary coproporphyria.

Received for publication, June 1, 2004 , and in revised form, June 10, 2004.

The atomic coordinates and structure factors (code 1tk1, 1tkl, and 1tlb) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by National Institutes of Health Grants GM56775 and DK20503. Operations of the National Synchrotron Light Source are supported by the United States Department of Energy, Office of Basic Energy Sciences, and by the National Institutes of Health. Data collection at the National Synchrotron Light Source was supported by the National Center for Research Resources. 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.

Both authors contributed equally to this work.

^¶ To whom correspondence may be addressed: Division of Hematology, 4C416 SOM, University of Utah School of Medicine, 50 N. 1900 E., Salt Lake City, UT 84132. Tel.: 801-581-6650; Fax: 801-585-5469; E-mail: john.phillips{at}hsc.utah.edu.

^¶¶ To whom correspondence may be addressed: Dept. of Biochemistry, University of Utah School of Medicine, Rm. 211, 20 N. 1900 E., Salt Lake City, UT 84132. Tel.: 801-585-5536; Fax: 801-581-7959; E-mail: chris{at}biochem.utah.edu.

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