Eukaryotic (THI4/THI5-type) de novo thiamine diphosphate biosynthesis

De novo synthesis of thiamine diphosphate (ThDP), the active vitamin B1 cofactor, by the route used in fungi. The thiazole and pyrimidine halves of thiamine are made separately and then joined. Both halves are made by single-turnover ("suicide") enzymes that donate part of their own structure to the product. The thiazole synthase THI4 builds an adenylated thiazole (ADP-5-ethyl-4-methylthiazole-2-carboxylate, ADT) from NAD+ and glycine, using the sulfur of one of its own active-site cysteines, which is left as a dehydroalanine; THI4 orthologues do the same in plants. The pyrimidine synthase THI5 (NMT1 in fission yeast) makes 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate (HMP-P) from pyridoxal 5'-phosphate and the side chain of one of its own histidine residues, in an iron-dependent reaction. ADT is converted to the thiazole phosphate by steps whose enzymes are still unknown, and HMP-P is phosphorylated to the diphosphate (HMP-PP) by HMP-P kinases of the ThiD family, which are fused to a TenA-like domain in budding yeast. The bifunctional THI6 protein joins the thiazole phosphate and HMP-PP to thiamine monophosphate; its second domain is a hydroxyethylthiazole kinase that can bring free thiazole into the pathway. Thiamine monophosphate is dephosphorylated to thiamine by a phosphatase that has not been identified, and thiamine pyrophosphokinase converts thiamine to ThDP. The enzymes are cytosolic. This route differs from the bacterial ThiC/ThiG pathway, and plants combine a THI4-type thiazole synthase with a bacterial-type ThiC pyrimidine synthase.

Boundary: the module runs from the two suicide synthases to ThDP. Thiamine uptake (THI7, THI72, NRT1; S. pombe thi9), mitochondrial ThDP import (TPC1; S. pombe SPBC1604.04, included in the PomBase model), the periplasmic thiamine phosphatase PHO3, and the THI regulon regulators are outside the module. The regulators are the transcription factors THI2 and PDC2 and the ThDP-sensing coactivator THI3 in S. cerevisiae (THI3 binds ThDP and is not an enzyme of the pathway), and thi1 and thi5 in S. pombe, which appear in the PomBase model as positive regulators of nmt1. The human uptake and activation pathway is in modules/thiamine_uptake_and_activation.yaml, and the bacterial ThiC/ThiG pathway is in modules/thiamine_diphosphate_biosynthesis.yaml. PLP for THI5 is consumed as a substrate, so this pathway depends on vitamin B6 supply (modules/vitamin_b6_plp_metabolism.yaml); in S. cerevisiae the thiamine- induced SNZ2/SNZ3-SNO2/SNO3 PLP synthase copies are linked to this demand (PMID:12271461). THI22, the third THI20-family paralogue, has no demonstrated HMP-P kinase activity and is dispensable for thiamine synthesis (see genes/yeast/THI22), so it is not used as an exemplar of the kinase step. The THI20 C-terminal TenA-like domain (thiaminase II) belongs to thiamine salvage and degradation, not to this module. Plants share the THI4 step (Arabidopsis THI1 is included as a representative) but make the pyrimidine with a bacterial-type ThiC and use a TH1 HMP-P kinase/TMP synthase fusion, so the plant pathway is not modelled as a full variant here. GO-CAM notes: the SGD model (no causal edges) lists THI13 as the only THI5-family enzyme, types THI20 and THI21 with both kinase activities, and lacks THI6 hydroxyethylthiazole kinase, the ADT conversion, and the thiamine phosphate phosphatase. The PomBase model includes the THI6 kinase activity and an unassigned thiamine phosphate phosphatase.