Structural studies of substrate and product complexes of 5-aminolaevulinic acid dehydratase from humans, Escherichia coli and the hyperthermophile Pyrobaculum calidifontis.

摘要: A number of X-ray analyses an enzyme involved in a key early stage tetrapyrrole biosynthesis are reported. Two structures human 5-aminolaevulinate dehydratase (ALAD), native and recombinant, have been determined at 2.8 A resolution, showing that the adopts octameric quaternary structure accord with previously published from range other species. However, this is contrast to finding disease-related F12L mutant uniquely forms hexamers [Breinig et al. (2003), Nature Struct. Biol. 10, 757–763]. Monomers all ALADs adopt TIM-barrel fold; subunit conformation assembles into octamer includes N-terminal tail one monomer curled around (α/β)8 barrel neighbouring monomer. Both crystal possess two monomers per asymmetric unit, termed B. In there distinct structural differences between B monomers, latter exhibiting greater disorder loop regions active site. contrast, second recombinant appears be better defined site both clearly possesses zinc ion which bound by three conserved cysteine residues. ALAD, also has ligand resembling substrate ALA covalently Schiff base active-site lysines (Lys252) held place ordered loop. these features disordered or absent enzyme. The zinc-dependent ALAD hyperthermophile Pyrobaculum calidifontis reported somewhat lower resolution 3.5 A. Finally, details presented high-resolution Escherichia coli co-crystallized noncovalently moiety product, porphobilinogen (PBG). This reveals pyrrole side-chain amino group datively PBG carboxylates interact via hydrogen bonds salt bridges invariant hydrogen-bond interactions were observed yeast cyclic intermediate product appear weaker new structure, suggesting only optimal transition state.