Folate biosynthesis and structures of pathway intermediates

Folic acid, also known as Vitamin B9 is important to several biological functions. The folate derivative, 5,10-methylene-tetrahydrofolate is essential for the synthesis of dTMP from dUMP and it is therefore crucial for DNA replication and cell division. Tetrahydrofolate is an essential substrate in the biosynthesis of amino acid, glycine. Drugs targeting folate biosynthesis pathway has long been prescribed as anti-malarial agents. The two essential precursors of folate biosynthesis are 4-aminobenzoate (a product of pathway) and GTP. Thymidylate cycle, a part of folate biosynthesis pathway (below) plays important role in the generation of amino acid glycine and dTMP. Dihydrofolate reductase enzyme replenishes tetrahydrofolate from dihydrofolate for the above mentioned biosynthetic processes. The dihydrofolate reductase and thymidylate synthase activities are catalysed by a bifunctional enzyme in both Plasmodium falciparum and Toxoplasma gondii. In addition to the de novo folate biosynthesis pathway, T. gondii can salvage folate from host. Massimine et al demonstrated the uptake of radio-labelled exogenous folic acid and revealed the presence of common folate transporter which has high affinity for folic acid. This transporter is suggested to be bidirectional and concentration-dependent. They also added that T. gondii and other apicomplexans encode folate transporters as there are putative transporters homologous to BT1 family proteins present in these Apicomplexa genomes [].

(a) Conventional and alternative folate biosynthetic pathways

Besides the de novo biosynthesis of BH4, SR is also known to be involved in the pterin salvage pathway catalysing the conversion of sepiapterin to dihydrobiopterin (BH2) which is transformed by dihydrofolate reductase to BH4.

Biosynthetic pathways using tetrahydrofolate and …

And what of ? Recall that was the only gene to be successfully cloned into an inducible-copy plasmid, and so it was tested both induced and uninduced. The folate levels in the induced sample (high-copy) are higher than in the uninduced (low-copy) sample, which is consistent with what we would expect. The addition of PABA to both induced and uninduced increases relative levels of folate, which is also consistent. However, it is interesting to note that folate levels for the +PABA samples are the same for induced and uninduced. Of course, given the small sample size, this could just be due to variability, but it could also suggest that folate production reached an upper limit. This explanation seems even more likely if we reconsider the folate biosynthesis pathway, and we see that and are both located upstream of actual integration of PABA into the molecule, accomplished by . It is very possible that we are increasing the flux of both pathways going into the junction, but as we were unable to overexpress as well, we may have created a bottleneck.