Abstract
Uroporphyrinogen III, the universal progenitor of macrocyclic, modified tetrapyrroles, is produced from aminolaevulinic acid (ALA) by a conserved pathway involving three enzymes: porphobilinogen synthase (PBGS), hydroxymethylbilane synthase (HmbS) and uroporphyrinogen III synthase (UroS). The gene encoding uroporphyrinogen III synthase has not yet been identified in Plasmodium falciparum, but it has been suggested that this activity is housed inside a bifunctional hybroxymethylbilane synthase (HmbS). Additionally, an unknown protein encoded by PF3D7_1247600 has also been predicted to possess UroS activity. In this study it is demonstrated that neither of these proteins possess UroS activity and the real UroS remains to be identified. This was demonstrated by the failure of codon-optimized genes to complement a defined Escherichia coli hemD − mutant (SASZ31) deficient in UroS activity. Furthermore, HPLC analysis of the oxidized reaction product from recombinant, purified P. falciparum HmbS showed that only uroporphyrin I could be detected (corresponding to hydroxymethylbilane production). No uroporphyrin III was detected, showing that P. falciparum HmbS does not have UroS activity and can only catalyze the formation of hydroxymethylbilane from porphobilinogen.
Highlights
Haem, as an iron-containing porphyrin, is a modified tetrapyrrole that is derived from the starting material 5-aminolevulinic acid (5-aminolaevulinic acid (ALA)) [1]
To test if either P. falciparum hemC or P. falciparum 3D7_1247600 harbour uroporphyrinogen III synthase (UroS) activity, complementation studies were performed to see if either gene could restore growth to a defined hemD− mutant (SASZ31) lacking UroS activity [23]
The controls included an empty pET-3a as a negative control and plasmids harbouring known hemD genes from Bacillus megaterium and E. coli as positive controls
Summary
As an iron-containing porphyrin, is a modified tetrapyrrole that is derived from the starting material 5-aminolevulinic acid (5-ALA) [1]. The bilane undergoes cyclization, but only after inversion of the terminal D ring, to give uroporphyrinogen III [6, 7] These three steps are found in all organisms that make modified tetrapyrroles [1]. The evidence presented for this was HPLC identification of the (oxidized) reaction product as uroporphyrin III from both native and recombinant HmbS when incubated with PBG Such dual activity has previously been reported for HmbS from Leptospira interrogans, this is a very different protein from P. falciparum HmbS, being a fusion of HmbS and UroS enzymes [22]. A multiple sequence alignment is shown in Figs S1 and S2 (available in the online version of this article) It is, hard to understand how this enzyme could house two very different activities. The possibility that UroS activity is encoded by PF3D7_1247600 is investigated
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