Plasmodium falciparum hydroxymethylbilane synthase does not house any cosynthase activity within the haem biosynthetic pathway

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.

Revealing the promoting effect of betaine on vitamin B12 biosynthetic pathway of Pseudomonas denitrificans by using a proteomics analysis

Background: Our previous comparative metabolomics research revealed that betaine (N,N,N-trimethylglycine, a typically essential methyl-group donor for vitamin B12 biosynthesis) had a powerful promoting effect on the generation of vitamin B12 precursors and intermediates in vitamin B12-producing Pseudomonas denitrificans. However, the integral effect of betaine on the vitamin B12 biosynthetic pathway is still unclear. Objective: Considering the vitamin B12 biosynthetic pathway of P. denitrificans as a whole, this work aimed to reveal the biological function of betaine on the vitamin B12 biosynthetic pathway in P. denitrificans, which would sharpen and expand the understanding of betaine as the methyl-group donor for vitamin B12 biosynthesis. Materials and Methods: By using a proteomics method based on the iTRAQ technique, the present study compared and analyzed the differential expression of proteins involved in vitamin B12 biosynthetic pathway under 10 g/L betaine addition to P. denitrificans fermentation medium. Results: The results showed that betaine could significantly up-regulate the expression of proteins related to the vitamin B12 biosynthetic pathway, which was mainly reflected in the following three aspects: 1) the δ-aminolevulinic acid (ALA) synthase and porphobilinogen synthase that were responsible for the formation of the committed precursors for tetrapyrrole-derived macrocycle in vitamin B12 molecule; 2) the C-methylation-related enzymes (such as precorrin-4 C(11)-methyltransferase, Precorrin-2 C(20)-methyltransferase, Precorrin-8X methylmutase, and Precorrin-6Y C5,15-methyltransferase) and methionine synthase that were crucial to the C-methylation reactions for vitamin B12 biosynthesis; 3) the late-stage key enzymes (Cobaltochelatase, and Cob(I)yrinic acid a,c-diamide adenosyltransferase) that were related to cobalt chelation of vitamin B12 molecule. Conclusions: The present study clearly demonstrated that betaine could significantly promote the expression of the integral enzymes involved in the vitamin B12 biosynthetic pathway of P. denitrificans, thus promoting vitamin B12 biosynthesis.

Exploring human porphobilinogen synthase metalloprotein by quantum biochemistry and evolutionary methods

Previous studies have shown the porphobilinogen synthase (PBGS) zinc-binding mechanism and its conservation among the living cells. However, the precise molecular interaction of zinc with the active center of the enzyme is unknown. In particular, quantum chemistry techniques within the density functional theory (DFT) framework have been the key methodology to describe metalloproteins, when one is looking for a compromise between accuracy and computational feasibility. Considering this, we used DFT-based models within the molecular fractionation with conjugate caps scheme to evaluate the binding energy features of zinc interacting with the human PBGS. Besides, phylogenetic and clustering analyses were successfully employed in extracting useful information from protein sequences to identify groups of conserved residues that build the ions-binding site. Our results also report a conservative assessment of the relevant amino acids, as well as the benchmark analysis of the calculation models used. The most relevant intermolecular interactions in Zn2+–PBGS are due to the amino acids CYS0122, CYS0124, CYS0132, ASP0169, SER0168, ARG0221, HIS0131, ASP0120, GLY0133, VAL0121, ARG0209, and ARG0174. Among these residues, we highlighted ASP0120, GLY0133, HIS0131, SER0168, and ARG0209 by co-occurring in all clusters generated by unsupervised clustering analysis. On the other hand, the triple cysteines at 2.5 Å from zinc (CYS0122, CYS0124, and CYS0132) have the highest energy attraction and are absent in the taxa Viridiplantae, Sar, Rhodophyta, and some Bacteria. Additionally, the performance of the DFT-based models shows that the processing time-dependence is more associated with the choice of the basis set than the exchange–correlation functional.

Plasmodium falciparum hydroxymethylbilane synthase does not house any cosynthase activity within the haem biosynthetic pathway

The production of 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). In this present study it is demonstrated that P. falciparum HmbS does not have uroporphyrinogen III synthase activity. This was demonstrated by the failure of a codon optimised P. falciparum hemC gene, encoding HmbS, to compliment a defined E. coli hemD- mutant (SASZ31) deficient in uroporphyrinogen III synthase activity. Furthermore, HPLC analysis of the oxidsed reaction product from recombinant, purified HmbS showed that only uroporphyrin I could be detected (corresponding to hydroxymethylbilane production). No uroporphyrin III was detected, thus showing that P. falciparum HmbS does not have UroS activity and can only catalyse the formation of hydroxymethylbilane from porphobilinogen.