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.