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Studies on recombinant human 5-aminolaevulinic acid dehydratase and recombinant human porphobilinogen deaminase

Studies on recombinant human 5-aminolaevulinic acid dehydratase and recombinant human porphobilinogen deaminase
Studies on recombinant human 5-aminolaevulinic acid dehydratase and recombinant human porphobilinogen deaminase

5-Aminolaevulinic acid dehydratase (ALAD) catalyses the condensation between two molecules of 5-aminolaevulinic acid to form porphobilinogen (PBG). Recombinant human ALAD, encoded by a synthetic gene, has been over-expressed and purified to yield milligram amounts of enzyme for characterisation.  The recombinant enzyme was crystallised and the structure revealed a ligand resembling the product, PBG, at the active sites. Experiments described in this thesis indicate the bound ligand is not porphobilinogen but a putative intermediate, which has provided clues to the enzymes mechanism.

The human ALAD mutant, Phe12Leu, has been characterised. Phenylalanine 12 is located on the N-terminal arm and is thought to be involved in subunit interactions. Hybrid octamers of the mutant and the normal ALAD were formed, identifying residues that may be important in communication between subunits.

Porphobilinogen deaminase (PBGD) polymerises PBG to form the tetrapyrrole preuroporphyrinogen in the third step of the haem biosynthesis pathway. Recombinant erythroid and ubiguitous human PBGD were purified from an engineered strain of E. coli. The purified PBGD appeared homogeneous when analysed by SDS-PAGE but existed in the form of a distinct double band when analysed by non-denaturing PAGE.  Proteolytic digestion and peptide analysis of the two separated deaminase species was carried out to identify differences between the two protein forms.

A number of PBGD mutants were studied and some were found to accumulate enzyme substrate (ES) complexes. This thesis describes the purification of these complexes and their attempted crystallisation as the structure of an enzyme substrate complex would provide a wealth of information on the enzyme mechanism.

University of Southampton
Butler, Danica Simone
Butler, Danica Simone

Butler, Danica Simone (2003) Studies on recombinant human 5-aminolaevulinic acid dehydratase and recombinant human porphobilinogen deaminase. University of Southampton, Doctoral Thesis.

Record type: Thesis (Doctoral)

Abstract

5-Aminolaevulinic acid dehydratase (ALAD) catalyses the condensation between two molecules of 5-aminolaevulinic acid to form porphobilinogen (PBG). Recombinant human ALAD, encoded by a synthetic gene, has been over-expressed and purified to yield milligram amounts of enzyme for characterisation.  The recombinant enzyme was crystallised and the structure revealed a ligand resembling the product, PBG, at the active sites. Experiments described in this thesis indicate the bound ligand is not porphobilinogen but a putative intermediate, which has provided clues to the enzymes mechanism.

The human ALAD mutant, Phe12Leu, has been characterised. Phenylalanine 12 is located on the N-terminal arm and is thought to be involved in subunit interactions. Hybrid octamers of the mutant and the normal ALAD were formed, identifying residues that may be important in communication between subunits.

Porphobilinogen deaminase (PBGD) polymerises PBG to form the tetrapyrrole preuroporphyrinogen in the third step of the haem biosynthesis pathway. Recombinant erythroid and ubiguitous human PBGD were purified from an engineered strain of E. coli. The purified PBGD appeared homogeneous when analysed by SDS-PAGE but existed in the form of a distinct double band when analysed by non-denaturing PAGE.  Proteolytic digestion and peptide analysis of the two separated deaminase species was carried out to identify differences between the two protein forms.

A number of PBGD mutants were studied and some were found to accumulate enzyme substrate (ES) complexes. This thesis describes the purification of these complexes and their attempted crystallisation as the structure of an enzyme substrate complex would provide a wealth of information on the enzyme mechanism.

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Published date: 2003

Identifiers

Local EPrints ID: 465270
URI: http://eprints.soton.ac.uk/id/eprint/465270
PURE UUID: c227d960-f7fd-4d09-8816-36d1c5b4f398

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Date deposited: 05 Jul 2022 00:34
Last modified: 05 Jul 2022 04:45

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Author: Danica Simone Butler

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