Structural characterisation of the active site of escherichia coli 5-aminolaevulinic acid dehydratase

Abstract

5-Aminolaevulinic acid dehydratase (ALAD) is an early enzyme in the biosynthesis pathway of catalysing the condensation between two 5-aminolaevulinic acid (ALA) molecules to form porphobilinogen (PEG), the monopyrrole precursor of all tetrapyrroles. Within the active site of E. coli ALAD there are two discrete substrate binding sites. The first substrate molecule to bind to the enzyme forms the propionate half of PEG whilst the second ALA molecule forms the acetate half, leading to the terminology of "P" and "A" ALA binding sites. This thesis describes the co-crystallisation and structural determination of E. coli ALAD with ALA present at both the P- and A-sites allowing extensive characterisation of substrate binding residues. These observations are confirmed by the crystal structure of ALAD in complex with the product, PEG. Site directed mutagenesis of residues implicated in substrate binding and catalysis by these structural studies has been carried out, and the contribution of each residue to PEG formation determined. Two invariant P- site lysine residues, K247 and K195, were the subject of further study. Mutants K247A and K247C proved to be completely inactive whereas mutants K195A and K195C retained < 0.1% of wild type activity. Activity could be rescued by modification of K247C and K195C to lysine analogues with bromoethylamine. Low levels of activity shown by the mutant K247N are explained by its crystal structure in complex with laevulinic acid. The pKg of Lys 247 is shown to be suppressed to 7.5 by Lys 195, allowing it to form a Schiff base with substrate ALA at physiological pH values. E. coli ALAD in complex with the inhibitor molecule 5-chlorolaevulinic acid has been crystallised and the structure determined, revealing alkylation of Lys 195. Conclusions are drawn regarding the contributions of active site residues to the mechanism of PEG formation. 11

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