CRYSTALLOGRAPHIC STUDIES ON THE BIFUNCTIONAL PTERIN-4A-CARBINOLAMINE DEHYDRATASES FROM HUMAN LIVER AND PSEUDOMONAS AERUGINOSA. Dietrich Suck, Ralf Ficner, Uwe H. Sauer, Gunter Stier, EMBL, Meyerhostrasse 1, 69117 Heidelberg, Germany

The bifunctional protein pterin-4a-carbinolamine dehydratase (PCD) is a cytoplasmic enzyme involved in the regeneration of tetrahydrobiopterin, an essential cofactor of several monooxygenases. PCD is also found in cell nuclei forming a tight complex with the transcription factor HNF1. PCD binds to the dimerization domain of HNF1 and accordingly it is called dimerization cofactor of HNF1 (DCoH) as well. The functional enzyme PCD is a homotetramer while it interacts as a dimer with the dimeric HNF1.

The crystal structure of tetrameric PCD/DCoH from rat/human liver was solved by MIR and refined to a R-factor of 20.5% at 2.7 Å resolution (1). The single domain monomer (12 kDa) comprises three [[alpha]]-helices packed against one side of a four-stranded, antiparallel [[beta]]-sheet. The homotetramer displays 222 symmetry and can be viewed as a dimer of dimers. In the dimer two monomers form an eight-stranded, antiparallel [[beta]]-sheet with all helices packing against it on one side. In the tetramer the interface between both dimers is a central four helix bundle where each of the monomers contributes one helix to it. The concave, hydrophobic surface of the eightstranded [[beta]]-sheet of the dimers is reminiscent of the saddle like shape seen in the TATA-box binding protein.

Recently, a bacterial homologue of PCD/DCoH, called PhhB, was found in Pseudomonas aeruginosa showing a dehydratase activity similar to the mammalian PCD. This procaryotic PCD is also bifunctional, as it regulates the expression of the P. areuginosa phenylalanine hydroxylase gene.

Here we present the overexpression, purification, and crystallization of the procaryotic PCD. The crystal structure was solved by means of MAD using seleno-methionine modified PCD and the refinement is currently in progress. The comparison of the mammalian PCD structure with the bacterial one, and preliminary results of mutational studies provide insight into the catalytic mechanism.

(1) Ficner, R., Sauer, U. H., Stier, G. and Suck, D. (1995) EMBO J. 14, 2034-2042.