X-ray structure of 5-aminolevulinic acid dehydratase from Escherichia colicomplexed with the inhibitor levulinic acid at 2.0 angstrom resolution

5-Aminolevulinic acid dehydratase (ALAD), an early enzyme of the tetrapyrro le biosynthesis pathway, catalyzes the dimerization of 5-aminolevulinic aci d to form the pyrrole, porphobilinogen, ALAD from Escherichia coli is shown to form a homo-octameric structure with 422 symmetry in which each subunit adopts the TIM barrel fold with a 30-residue N-terminal arm. Pairs of mono mers associate with their arms wrapped around each other. Four of these dim ers interact, principally via their arm regions, to form octamers in-which each active site is located on the surface. The active site contains two ly sine residues (195 and 247), one of which (Lys 247) forms a Schiff base lin k with the bound substrate analogue, levulinic acid, Of the two substrate b inding sites (referred to as A and P), our analysis defines the residues fo rming the P-site, which is where the first ALA molecule to associate with t he enzyme binds. The carboxyl group of the levulinic acid moiety forms hydr ogen bonds with the side chains of Ser 273 and Tyr 312, In proximity to the levulinic acid is a zinc binding site formed by three cysteines (Cys 120, 122, and 130) and a solvent molecule. We infer that the second substrate bi nding site (or A-site) is located between the triple-cysteine zinc site and the bound levulinic acid moiety. Two invariant arginine residues in a loop covering the active site (Arg 205 and Arg 216) appear to be appropriately placed to bind the carboxylate of the A-site substrate. Another metal bindi ng site, close to the active site flap, in which a putative zinc ion is coo rdinated by a carboxyl and five solvent molecules may account for the activ ating properties of magnesium ions.