Studies on recombinant P. Sativum and human 5-aminolaevulinic acid dehydratases

Abstract

5-Aminolaevulinic acid dehydratase catalyses the dimerisation of two molecules of 5-aminolaevulinic acid to form the pyrrole, porphobilinogen.  This is the first common step in the biosynthesis of haems, chlorophylls, corrins and other tetrapyrroles.  A recombinant form of 5-aminolaevulinic acid dehydratase from P. sativum¸ that lacks the N-terminal chloroplast transit peptide, has been overexpressed in Escherichia coli and isolated by the use of a novel purification technique.  This has yielded milligram amounts of enzyme that have enabled the characterization and crystallisation of the enzyme to be accomplished.  Experiments indicate the possibility of alternative quaternary forms of the enzyme that may be modulated by pH, temperature and enzyme concentration.  Quantitative investigations into the nature of the chemical dimerisation of the substrate 5-aminolaevulinic acid to the dihydropyrazine have also been investigated under different assay buffer conditions and the implications on the activity of P. sativum, human and E.coli 5-aminolaevulinicacid dehydratases have been compared.

Purified P. sativum 5-aminolaevulinic acid dehydratase yielded crystals that diffract to 2.9Å and an adapted selenomethionine growth and purification protocol has yielded a selenomethionine form of the enzyme.  Despite extensive attempts, using a variety of programmes, a crystal structure has not yet been solved and studies are continuing.

The structure of human recombinant erythrocyte 5-aminolaevulinic acid dehydratase has been solved and compared to that of the native species.  The presence of a putative intermediate that is tightly bound at the active site and has given further insight into the enzyme mechanism.

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