THE X-RAY STRUCTURES OF 5-AMINOLAEVULINATE DEHYDRATASE FROM E. COLI AND YEAST.

Peter T Erskine¹, Richard Newbold², Alun Coker¹, Gareth Lewis¹, Martin J Warren², Peter M Shoolingin-Jordan¹, Steve P Wood¹ and Jon B Cooper¹

¹Division of Biochemistry and Molecular Biology, School of Biological Sciences, University of Southampton, Bassett Crescent East, Southampton, SO16 7PX, UK.
² Department of Molecular Genetics, Institute of Ophthalmology, University College London, Bath Street, London, EC1V 9EL, UK.

Background: 5-aminolaevulinate dehydratase (ALAD, porphobilinogen synthase) is a key early enzyme of the porphyrin and corrin biosynthetic pathways which catalyses the condensation of two 5-aminolaevulinic acid (ALA) molecules to form the pyrrole porphobilinogen (PBG). The hereditary deficiency of functional dehydratase in humans is associated with the genetic disease Doss or ALA dehydratase porphyria, a disease with severe neurological symptoms. ALAD is extremely sensitive to inhibition by lead ions which is one of the major manifestations of acute lead poisoning which often leads to neurological disturbances. ALAD has recently been identified as a putative regulatory component of the eukaryotic 26S proteasome.

Results: The yeast ALAD structure (solved at 2.3 Ä by selenomethionine MAD at ESRF, Grenoble with Dr A. Thompson [1]) was used to solve the E. coli enzyme at 2.0 Ä by molecular replacement. ALAD from both species forms a large homo-octameric structure with 422 symmetry in which each subunit adopts the (a/b)₈ or TIM-barrel fold with a 20-30 residue N-terminal arm forming extensive inter-subunit interactions. Pairs of monomers associate with their arms wrapped around each other to form compact dimers. Four dimers interact principally via their arm regions to form the octamer which has all eight active sites exposed on the surface. At the base of each active site are two lysine residues (195 and 247 in E. coli numbering), one of which, Lys 247, forms a Schiff base link to the substrate ALA. Close by is a zinc binding site formed by three cysteines (Cys 120, 122 and 130) and a solvent molecule. The structure of the inhibitor laevulinic acid bound to both ALADs has been analyed at high resolution as has the structure of the substrate ALA bound to the enzyme's P-site.

Conclusions: A large loop covering the active site (residues 197-222), much of which was completely disordered in the native yeast ALAD structure, undergoes a substantial ordering upon binding of the laevulinic acid inhibitor. The P-site, which binds the first substrate molecule to dock with the enzyme in its catalytic cycle, is defined by the interactions observed in these complexes. The second substrate moiety to bind does so at the enzyme's more elusive A-site which is presumed to involve the zinc ion held by three cysteines. The E. coli enzyme possesses another well defined zinc binding site in which the metal ion is coordinated by the carboxyl of Glu 232 and five solvent molecules buried at a subunit interface. This site is in a water-filled pocket adjacent to the Schiff base lysine. The discovery of this octahedrally coordinated metal binding site and its proximity to the active site may account for the activating properties which magnesium ions have on the enzyme.

[1] Erskine, P.T. et al., Nature Structural Biology 4(1997) 1025-1031.