Studies on the uroporphyrinogen decarboxylase and corproporphyinogen III oxidase of the photosynthetic bacterium, Rhodobacter sphaeroides

Jones, Russell Mark (1992) Studies on the uroporphyrinogen decarboxylase and corproporphyinogen III oxidase of the photosynthetic bacterium, Rhodobacter sphaeroides. University of Southampton, Doctoral Thesis.

Record type: Thesis (Doctoral)

Abstract

Uroporphyrinogen decarboxylase and coproporphyrinogen III oxidase were studied in the photosynthetic bacterium, Rhodobacter sphaeroides. Uroporphyrinogen decarboxylase was purified, 600 fold, to homogeneity in yields of 40%. Uroporphyrinogen decarboxylase was found to be a monomer of Mr 41,000, both under denaturing and non-denaturing conditions. Analysis of the absorbtion spectrum of the enzyme gave a single peak at 280 nm with no evidence for any chromomorphic cofactors, while determination of the pH optimum and isoelectric point revealed values of 6.8 and 4.3, respectively. Kinetic studies have revealed a K_m of 1.8 μM and 6.0 μM for the conversion of uroporphyrinogen I and III, to coproporphyrinogen I and III, respectively. The enzyme was susceptible to inhibition by its substrates at high concentrations and by the presence of porphyrins, especially the oxidised product, coproporphyrin III. Further investigations have shown the enzyme to be inhibited by metal ions, particularly mercury, which implicate the involvement of a cysteine residue. This observation is confirmed by the inhibition of activity by sulphydryl reagents. Titration of cysteine residues with DTNB has revealed five residues, one of which has been shown to be important for the activity of the enzyme. Arginine and to a lesser extent lysine residues have also been implicated in activity. Uroporphyrinogen decarboxylase was found to be inactivated at temperatures above 45^oC. The amino acid sequence for the first thirty residues of the N-terminus have been determined. The pathway of decarboxylation of uroporphyrinogen III to coproporphyrinogen III by uroporphyrinogen decarboxylase was also investigated. A mutant of R. sphaeroides, N1, has been isolated by its inability to grow under conditions of limiting oxygen in the light, while growth under chemoheterotrophic conditions, was unaffected. The inability to grow under photosynthetic conditions was accompanied by the excretion of coproporphyrin III into the media. Analysis by h.p.l.c. and direct enzyme assay has determined that the N1 mutant contains a defective coproporphyrinogen III oxidase. The N1 locus, which contains an open reading frame of 1.2 kb encoding a polypeptide of 307 amino acids, has been isolated and cloned into the pUC18/19 expression system and has resulted in a 2.4 fold increase in coproporphyrinogen III oxidase activity in E. coli. It is therefore proposed that this locus contains the putative hemF gene, which encodes the anaerobic coproporphyrinogen III oxidase.

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