Mikula I.1, Bodnárová M.1, Lee D.-S.2, Flachsová E.1, Rosipal R.1, Zeman J.1, Moroz L.3, Raman C.S. 2, Martásek P.1
1CIG, Charles University, Prague, Czech Republic, 2University of Texas Medical School, Houston, TX, USA, 3University of Florida, FL, USA
Heme, iron(II)-protoporphyrin, is indispensable for life. The biosynthetic pathway for heme is conserved across all three phyla - bacteria, archaea, and eukarya. Heme acts as a prosthetic group for numerous essential hemoproteins. Genetic defects in the heme biosynthetic pathway results in disorders known as porphyrias. Coproporphyrinogen oxidase (CPO) catalyzes the antepenultimate step in heme biosynthesis. CPO deficiency causes hereditary coproporphyria (HC) (1), inherited in an autosomally dominant fashion. Although a decade has passed since the cloning of human CPO, structural insights into this very important enzyme has not been forthcoming due to difficulties with crystallization. To overcome these hurdles we have resorted to two surefire strategies: (a) utilizing disease-causing CPO variants and (b) cloning of CPO from a number of mesophilic and thermophilic bacteria. The basic idea behind this strategy is that it utilizes naturally occurring variants to increase the chances of obtaining diffraction quality crystals. Two naturally occurring CPO mutants (R331W and Δ390Gly) and CPO from Chloroflexus aurantiacus and Thermosynechococcus elongatus were chosen as lead candidates for our trials. Cloning and overexpression of these proteins in E. coli has allowed us to successfully narrow down crystallization conditions for one or more of these proteins. In addition, we have also cloned CPO from the sea slug Aplysia californica and will present its expression profile in this well-studied model organism. Examination of the CPO sequences from 47 species reveals a highly conserved motif, RRGRYVEFNL, that may play a crucial role in substrate binding and catalysis. Based on the available data, we have constructed a model to describe how CPO recognizes coproporphyrinogen III and converts it to protoporphyrinogen IX. Diffracting crystals and phase information have been obtained for human and bacterial CPO and structural refinement is in progress.[Supported by Grant from MSMT of Czech Republic (LN 00A079)] (1) Martásek, P. Semin. Liver Dis. 18: 25-32, 1998.