|Notes|| Thiamin monophosphate is formed by coupling of two independently synthesized moieties, hydroxymethylthiamin-PP (HMP-PP) and hydroxyethylthiazole-P (HET-P). The thiazole moiety is biosynthesized in Bacillus subtilis and most other bacteria from 1-deoxy-D-xylulose-5-phosphate (DXP), glycine, and cysteine in a complex oxidative condensation reaction . This reaction requires five different proteins, ThiO, ThiG, ThiS, ThiF, and a cysteine desulfurase. Glycine oxidase (ThiO) catalyzes the oxidation of glycine to the corresponding glycine imine. Sulfur carrier protein adenylyl transferase (ThiF) catalyzes the adenylation of the carboxy-terminus of the sulfur carrier protein (ThiS-carboxylate), and cysteine desulfurase catalyzes the transfer of sulfur from cysteine to the ThiS-acyl adenylate to give ThiS-thiocarboxylate. ThiG is the thiazole synthase and catalyzes the formation of the thiazole from dehydroglycine, DXP and ThiS-thiocarboxylate [2, 3, 4]. The thiazole moiety of thiamin in E. coli is derived from tyrosine, cysteine and 1-deoxy-D-xylulose phosphate . The conversion of 5-aminoimidazole ribonucleotide (AIR) into 4-amino-2-methyl-5-hydroxymethylpyrimidine (HMP) is a fascinating reaction on the thiamin biosynthetic pathway in bacteria and is probably the most complex unresolved rearrangement in primary metabolism. The thiC gene complements all HMP requiring mutants in E.coli, S.typhimurium ,and B.subtilis .
#1 ThiC: The pyrimidine moiety of thiamin, HMP-PP, is synthesized from 5-aminoimidazole ribonucleotide (AIR), an intermediate of the purine biosynthesis pathway.
#2,3,4,5,6 ThiG, ThiS, ThiF, ThiH (or ThiO): The thiazole moiety of thiamin in E. coli is derived from tyrosine, cysteine and 1-deoxy-D-xylulose phosphate. Instead of tyrosine, B. subtilis uses glycine as a substrate for the synthesis of thiazole. In E. coli, two of the proteins required for the biosynthesis of thiazole are ThiG and ThiH. Thiazole synthase ThiG catalyzes the formation of the thiazole phosphate ring. Function of ThiH is not yet known. B. subtilis has another ThiG-associated enzyme, the FAD-dependent glycine oxidase ThiO, which catalyzes the oxidation of glycine to the corresponding imine. The closest structural relatives of ThiO are sarcosine oxidase and d-amino acid oxidase. The ThiO structure in B. subtilis has been resolved. ThiF catalyzes adenylation of the sulfur carrier protein ThiS by ATP. Because of the similarities between the ThiF/ThiS and MoeB/MoaD proteins involved in the biosynthesis of thiamin and molybdopterin cofactor, respectively, the sulfur transfer chemistry of these two biosynthetic pathways can be shared in some bacteria with only one ThiF/MoeB homolog.
#7 THI4: The THI4 protein family has been shown to be involved in the thiazole synthesis in some eukaryotes  (Thi4 from S. cerevisiae, Thi2 from S. pombe, Thi1 from Zea mays, and STI35 from Fusarium sp). Among prokaryotes, Thi4 homologs are present in T. maritima and Archaea, replacing the bacterial-type thiazole synthesis pathway (#2,3,4,5,6).
#8 ThiD: HMP-P is phosphorylated by the bifunctional HMP kinase/HMP-P kinase ThiD.
#9 ThiN: ThiD from T. maritima and most archaea have an additional C-terminal domain of approximately 130 aa, named ThiN, whereas this domain is encoded by a separate gene in some Archaea. ThiN, is not similar to any known protein and contains no conserved motifs. In all cases when ThiE is absent and ThiD is present, there is the ThiN domain, although in many cases ThiN and ThiE co-exist. We suggest that this conserved domain is somehow involved in the TBS, possibly replacing the ThiE function in the genomes of some archaea and T. maritima.
#10 ThiM: The HET kinase ThiM is involved in the salvage of thiazole from the culture medium.
#11 ThiE: The ThiE protein catalyzes the formation of thiamin monophosphate via coupling of hydroxymethylthiamin-PP and hydroxyethylthiazole-P moieties.
#12 TenA: Conserved thiamin-related protein TenA is often associated with ThiD  (in some eukaryotes and bacteria tenA is fused to thiD). In systematic the discovery of analogous enzymes cariied out by Morett et al., 2003 , TenA have been shown to complement the thiC mutant in E. coli., however in most genomes the tenA and thiE genes co-exist. Recent biochemical characterization of TenA found the thiaminase II activity for this protein .
#13 ThiL: Thiamin monophosphate is phosphorylated by ThiL to form thiamin pyrophosphate.
#14, 15, 16 ThiXYZ: The hypothetical thiamin-related ABC transporter thiXYZ have upstream THI elements in all cases with only one exception in T. maritime . In addition the thiXYZ operons are often clustered with thiD. ThiXYZ was not found in the genomes without thiamin biosynthesis, but sometimes it occurs in genomes with the incomplete pathway without the ThiC enzyme. The need of HMP moiety for the thiamin biosynthesis is obvious. However, pathways other than thiamin, that could supply these compounds, are not known. The putative substrate-binding protein ThiY is similar to enzymes for the HMP biosynthesis from yeasts (Thi3 of S. pombe and Thi5 of S.cerevisiae). All found ThiY orthologs are predicted to have an N-terminal transmebrane segment, which is common for substrate-binding components of ABC transporters. Thus, we predict that ThiXYZ is a HMP transport system which substitutes for missing HMP biosynthesis in some bacteria.
#17 YuaJ: The single THI-regulated gene yuaJ (the B. subtilis name) was found in most genomes of the Bacillus/Clostridium group . It is always preceded by a THI element and is never clustered with TBS genes (only C. perfringes has two yuaJ paralogs, with and without an upstream THI element). YuaJ has six predicted transmembrane segments and is not similar to any known protein. yuaJ is the only thiamin-regulated gene in the complete genomes of Streptococcus mutans and Streptococcus pyogenes, which have no genes for the thiamin pathway. These observations strongly suggest that YuaJ is a thiamin transporter, which, in contrast to ThiBPQ, is obviously not ATP-dependent. In support of this prediction, the thiamin uptake in Bacillus cereus, which has yuaJ, is coupled to the proton movement .
#18,19,20 ThiBPQ: Well-characterized in enterobacteria ABC-type thiamin uptake transporter.
#21 CytX: CytX encodes a hypothetical transporter with 12 predicted TMSs, which is similar to the cytosine permease CodB from E. coli . Orthologs of this gene found in some diverse bacterial and archaeal species. In all cases, cytX either clusters with the thiamin genes, or has upstream THI elements, or both. Based on positional analysis and similarity to pyrimidine transporter, the new thiamin-related transporter CytX is most likely involved in the HMP transport.
#22 ThiW: The thiamin operons of some bacteria from the Bacillus/Clostridium group encode yet another thiamin-related transporter with 5 predicted transmembrane segments . This gene, named thiW, is not similar to any known protein and has no homologs in other genomes. It is always THI-regulated and located immediately upstream of the thiM gene in all cases. As ThiW seems to complement the absence of HET pathway in T. tengcongensis and S. pneumoniae we tentatively predict that ThiW is involved in transport of the thiazole moiety of thiamin in the above bacteria.
#23 ThiU: This gene from the Pasteurellaecae taxon is similar to transporters from the MFS family. In all cases thiU is clustered with the thiMDE genes and THI-regulated . H. influenzae and P. multocida lack both HMP and HET pathways. The former is accounted for by the ThiXYZ system (see above). This, together with positional analysis, suggests that ThiU is a thiazole transporter.
#24 ThiT: The archaeal THI elements were found upstream of two paralogous genes, named thiT1 and thiT2, in each of the three Thermoplasma genomes . These genes encode hypothetical transmembrane proteins with 9 predicted TMSs similar to transporters of the MFS family. Specificity of these transporters is not clear because of incompleteness of the thiamin pathways in thermoplasmas. However, based on the assumption that these transporters are the only thiamin-regulated genes in thermoplasmas, we propose their possible involvement in the thiamin transport.
#25,26,27,28 YkoEDCF: In some Gram-positive bacteria, we have found another thiamin-related ABC transporter, YkoE-YkoD-YkoC. It consists of two transmembrane components (YkoE and YkoC) and ATPase component (YkoD). We could not identify a substrate-binding component for this system. Similarly to thiXYZ, the ykoEDC genes always co-occur with the thiamin genes and are preceded by a THI element. They have been also found in genomes with the incomplete thiamin pathway. In B. subtilis, the first gene of the THI–regulated ykoFEDC operon is not similar to any known protein and has only one ortholog in Mesorhizobium loti, where it clusters with the above described candidate HMP-transporter, forming a THI-regulated cluster ykoFXYZ. Thus, the new ABC transport system YkoEDC is obviously thiamin-related, and most likely is involved in the HMP transport for thiamin. This prediction is based on positional clustering and on the following fact: when YkoEDC occurs in genomes lacking both HMP and HET pathways, there always is a candidate HET transporter, but not other HMP transporters .
#29,30,31 PnuT, Omr1, TPPK (tnr3): In two bacteria from the CFB group, B. fragilis and P. gingivalis, THI elements precede a hypothetical operon, named omr1-pnuT-tnr3. The omr1 gene is similar to TonB-dependent outer membrane receptors of Gram-negative bacteria that perform high-affinity binding and energy-dependent uptake of specific substrates into the periplasmic space. In another bacterium from the CFB group, Polaribacter filamentus, omr1 is a single gene which is preceded by a THI element. The pnuT gene encodes a hypothetical transporter which is similar to PnuC, N-ribosylnicotinamide transporters from enterobacteria. The PnuT proteins form a single branch on the phylogenetic tree of the PnuC family of transporters. The last gene of the omr1-pnuT-tnr3 operon is weakly similar to the C-terminal part of the thiamin pyrophosphokinase TNR3 from yeast S. pombe. Based on these data, we propose that the hypothetical THI-regulated omr1-pnuT-tnr3 operon could be involved in the thiamin salvage from the culture medium (transport and its subsequent phosphorylation up to thiamin pyrophosphate) . In confirmation, the pnuT-tnr3 operon of H. pylori forms a divergon with the thiamin-related gene tenA.
#32 YlmB: Another new gene of the thiamin regulon belongs to the argE/dapE/ACY1/CPG2/yscS family of metallopeptidases. The single ylmB gene in B. subtilis and the putative ylmB-tenA-thiXYZ operon in B. halodurans are preceded by THI elements, but no regulatory element was found upstream of the single ylmB gene in B. cereus.
#33 TK: The thiamin kinase activity is present in E. coli but corresponding gene is not yet characterized .
#34 TPP: The thiamin-phosphate phosphatase activity is present in yeast but corresponding gene is not yet characterized. Bacteria synthesize TPP via single phosphorylation of TP. Whereas eukaryote use distinct pathway to form an active coenzyme TPP: hydrolysis of TP to free thiamin is followed by pyrophosphorylation .
#35 THI5: In yeast and plants, the pyrimidine moiety of thiamin is synthesized using a distinct gene (nmt1 in Neurospora crassa, or THI5 in yeasts), and the initial substrates appear to be a histidine and pyridoxol-P [14, 15].
#36 THI10: Thiamin transporter in yeast.
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