Identification of the amino acid residues involved in the species-dependent differences in the pyridoxine transport function of SLC19A3.

Recent studies have shown that human solute carrier SLC19A3 (hSLC19A3) can transport pyridoxine (vitamin B6) in addition to thiamine (vitamin B1), its originally identified substrate, whereas rat and mouse orthologs of hSLC19A3 can transport thiamine but not pyridoxine. This finding implies that some amino acid residues required for pyridoxine transport, but not for thiamine transport, are specific to hSLC19A3. Here, we sought to identify these residues to help clarify the unique operational mechanism of SLC19A3 through analyses comparing hSLC19A3 and mouse Slc19a3 (mSlc19a3). For our analyses, hSLC19A3 mutants were prepared by replacing selected amino acid residues with their counterparts in mSlc19a3, and mSlc19a3 mutants were prepared by substituting selected residues with their hSLC19A3 counterparts. We assessed pyridoxine and thiamine transport by these mutants in transiently transfected human embryonic kidney 293 cells. Our analyses indicated that the hSLC19A3-specific amino acid residues of Gln86, Gly87, Ile91, Thr93, Trp94, Ser168, and Asn173 are critical for pyridoxine transport. These seven amino acid residues were found to be mostly conserved in the SLC19A3 orthologs that can transport pyridoxine but not in orthologs that are unable to transport pyridoxine. In addition, these residues were also found to be conserved in several SLC19A2 orthologs, including rat, mouse, and human orthologs, which were all found to effectively transport both pyridoxine and thiamine, exhibiting no species-dependent differences. Together, these findings provide a molecular basis for the unique functional characteristics of SLC19A3 and also of SLC19A2.

Competition between Different S-Components for the Shared Energy Coupling Factor Module in Energy Coupling Factor Transporters.

Energy coupling factor (ECF) transporters take up micronutrients in Bacteria and Archaea. They consist of a membrane-embedded S-component that provides substrate specificity and a three-subunit ECF module that couples ATP hydrolysis to transport. The S-components ThiT (for thiamin) and NiaX (for niacin) from Lactococcus lactis form complexes with the same ECF module. Here, we assayed the uptake of thiamin and niacin in Escherichia coli cells expressing the transporter genes. We demonstrate that the two different S-components compete for the ECF module, and that competition is more efficient in the presence of the transported substrate. The data suggest that binding and release of the S-components is a step in the transport cycle.

Salvage of the thiamin pyrimidine moiety by plant TenA proteins lacking an active-site cysteine.

The TenA protein family occurs in prokaryotes, plants and fungi; it has two subfamilies, one (TenA_C) having an active-site cysteine, the other (TenA_E) not. TenA_C proteins participate in thiamin salvage by hydrolysing the thiamin breakdown product amino-HMP (4-amino-5-aminomethyl-2-methylpyrimidine) to HMP (4-amino-5-hydroxymethyl-2-methylpyrimidine); the function of TenA_E proteins is unknown. Comparative analysis of prokaryote and plant genomes predicted that (i) TenA_E has a salvage role similar to, but not identical with, that of TenA_C and (ii) that TenA_E and TenA_C also have non-salvage roles since they occur in organisms that cannot make thiamin. Recombinant Arabidopsis and maize TenA_E proteins (At3g16990, GRMZM2G080501) hydrolysed amino-HMP to HMP and, far more actively, hydrolysed the N-formyl derivative of amino-HMP to amino-HMP. Ablating the At3g16990 gene in a line with a null mutation in the HMP biosynthesis gene ThiC prevented its rescue by amino-HMP. Ablating At3g16990 in the wild-type increased sensitivity to paraquat-induced oxidative stress; HMP overcame this increased sensitivity. Furthermore, the expression of TenA_E and ThiC genes in Arabidopsis and maize was inversely correlated. These results indicate that TenA_E proteins mediate amidohydrolase and aminohydrolase steps in the salvage of thiamin breakdown products. As such products can be toxic, TenA_E proteins may also pre-empt toxicity.

The role of thiamine in cancer: possible genetic and cellular signaling mechanisms.

The relationship between supplemental vitamins and various types of cancer has been the focus of recent investigation, and supplemental vitamins have been reported to modulate cancer rates. A significant association has been demonstrated between cancer and low levels of thiamine in the serum. Genetic studies have helped identify a number of factors that link thiamine to cancer, including the solute carrier transporter (SLC19) gene, transketolase, transcription factor p53, poly(ADP-ribose) polymerase-1 gene, and the reduced form of nicotinamide adenine dinucleotide phosphate. Thiamine supplementation may contribute to a high rate of tumor cell survival, proliferation and chemotherapy resistance. Thiamine has also been implicated in cancer through its effects on matrix metalloproteinases, prostaglandins, cyclooxygenase-2, reactive oxygen species, and nitric oxide synthase. However, some studies have suggested that thiamine may exhibit some antitumor effects. The role of thiamine in cancer is controversial. However, thiamine deficiency may occur in patients with cancer and cause serious disorders, including Wernicke's encephalopathy, that require parenteral thiamine supplementation. A very high dose of thiamine produces a growth-inhibitory effect in cancer. Therefore, further investigations of thiamine in cancer are needed to clarify this relationship.

High-throughput screening of small molecules identifies hepcidin antagonists.

Anemia of inflammation (AI) is common in patients with infection, autoimmune diseases, cancer, and chronic kidney disease. Unless the underlying condition can be reversed, treatment options are limited to erythropoiesis-stimulating agents with or without intravenous iron therapy, modalities that are not always effective and can cause serious adverse effects. Hepcidin, the iron regulatory hormone, has been identified as a pathogenic factor in the development of AI. To explore new therapeutic options for AI and other iron-related disorders caused by hepcidin excess, we developed a cell-based screen to identify hepcidin antagonists. Of the 70,000 small molecules in the library, we identified 14 compounds that antagonized the hepcidin effect on ferroportin. One of these was fursultiamine, a Food and Drug Administration (FDA)-approved thiamine derivative. Fursultiamine directly interfered with hepcidin binding to its receptor, ferroportin, by blocking ferroportin C326 thiol residue essential for hepcidin binding. Consequently, fursultiamine prevented hepcidin-induced ferroportin ubiquitination, endocytosis, and degradation in vitro and allowed continuous cellular iron export despite the presence of hepcidin, with IC(50) in the submicromolar range. Thiamine, the fursultiamine metabolite, and benfotiamine, another thiamine derivative, did not interfere with the effect of hepcidin on ferroportin. Other FDA-approved thiol-reactive compounds were at least 1000-fold less potent than fursultiamine in antagonizing hepcidin. In vivo, fursultiamine did not reproducibly antagonize the effect of hepcidin on serum iron, likely because of its rapid conversion to inactive metabolites. Fursultiamine is a unique antagonist of hepcidin in vitro that could serve as a template for the development of drug candidates that inhibit the hepcidin-ferroportin interaction.

Vitamin B1 biosynthesis in plants requires the essential iron sulfur cluster protein, THIC.

Vitamin B1 (thiamin) is an essential compound in all organisms acting as a cofactor in key metabolic reactions and has furthermore been implicated in responses to DNA damage and pathogen attack in plants. Despite the fact that it was discovered almost a century ago and deficiency is a widespread health problem, much remains to be deciphered about its biosynthesis. The vitamin is composed of a thiazole and pyrimidine heterocycle, which can be synthesized by prokaryotes, fungi, and plants. Plants are the major source of the vitamin in the human diet, yet little is known about the biosynthesis of the compound therein. In particular, it has never been verified whether the pyrimidine heterocycle is derived from purine biosynthesis through the action of the THIC protein as in bacteria, rather than vitamin B6 and histidine as demonstrated for fungi. Here, we identify a homolog of THIC in Arabidopsis and demonstrate its essentiality not only for vitamin B1 biosynthesis, but also plant viability. This step takes place in the chloroplast and appears to be regulated at several levels, including through the presence of a riboswitch in the 3'-untranslated region of THIC. Strong evidence is provided for the involvement of an iron-sulfur cluster in the remarkable chemical rearrangement reaction catalyzed by the THIC protein for which there is no chemical precedent. The results suggest that vitamin B1 biosynthesis in plants is in fact more similar to prokaryotic counterparts and that the THIC protein is likely to be the key regulatory protein in the pathway.

Isolation and characterization of new thiamine-deregulated mutants of Bacillus subtilis.

In bacteria, thiamine pyrophosphate (TPP) is an essential cofactor that is synthesized de novo. Thiamine, however, is not an intermediate in the biosynthetic pathway but is salvaged from the environment and phosphorylated to TPP. We have isolated and characterized new mutants of Bacillus subtilis that deregulate thiamine biosynthesis and affect the export of thiamine products from the cell. Deletion of the ydiA gene, which shows significant similarity to the thiamine monophosphate kinase gene of Escherichia coli (thiL), did not generate the expected thiamine auxotroph but instead generated a thiamine bradytroph that grew to near-wild-type levels on minimal medium. From this DeltathiL deletion mutant, two additional ethyl methanesulfonate-induced mutants that derepressed the expression of a thiC-lacZ transcriptional reporter were isolated. One mutant, Tx1, contained a nonsense mutation within the B. subtilis yloS (thiN) gene that encodes a thiamine pyrophosphokinase, a result which confirmed that B. subtilis contains a single-step, yeast-like thiamine-to-TPP pathway in addition to the bacterial TPP de novo pathway. A second mutant, strain Tx26, was shown to contain two lesions. Genetic mapping and DNA sequencing indicated that the first mutation affected yuaJ, which encodes a thiamine permease. The second mutation was located within the ykoD cistron of the ykoFEDC operon, which putatively encodes the ATPase component of a unique thiamine-related ABC transporter. Genetic and microarray studies indicated that both the mutant yuaJ and ykoD genes were required for the derepression of thiamine-regulated genes. Moreover, the combination of the four mutations (the DeltathiL, thiN, yuaJ, and ykoD mutations) into a single strain significantly increased the production and excretion of thiamine products into the culture medium. These results are consistent with the proposed "riboswitch" mechanism of thiamine gene regulation (W. C. Winkler, A. Nahvi, and R. R. Breaker, Nature 419:952-956, 2002).

Biosynthesis of the thiazole moiety of thiamin pyrophosphate (vitamin B1).

While most of the proteins required for the biosynthesis of thiamin pyrophosphate have been known for more than a decade, the reconstitution of this biosynthesis in a defined biochemical system has been difficult due to the novelty of the chemistry involved. Here we demonstrate the first successful enzymatic synthesis of the thiazole moiety of thiamin from glycine, cysteine, and deoxy-D-xylulose-5-phosphate using overexpressed Bacillus subtilis ThiF, ThiS, ThiO, ThiG, and a NifS-like protein. This has facilitated the identification of the biochemical function of each of the proteins involved: ThiF catalyzes the adenylation of ThiS; NifS catalyzes the transfer of sulfur from cysteine to the acyl adenylate of ThiS; ThiO catalyzes the oxidation of glycine to the corresponding imine; and ThiG catalyzes the formation of the thiazole phosphate ring. The complex oxidative cyclization reaction involved in the biosynthesis of the thiamin thiazole has been greatly simplified by replacing ThiF, ThiS, ThiO, and NifS with defined biosynthetic intermediates in a reaction where ThiG is the only required enzyme.

Novel point mutation in the leucine-rich motif of the platelet glycoprotein IX associated with Bernard-Soulier syndrome.

Bernard-Soulier syndrome (BSS) is a rare inherited bleeding disorder which is caused by a qualitative or quantitative abnormality of the platelet glycoprotein (GP) Ib/IX/V complex. We examined a patient with BSS to find a molecular basis for the defect underlying this disease. The propositus was a 39-year-old Japanese female with life-long bleeding diathesis. Sequence analysis of the GPIX gene revealed a T-->C point mutation at nucleotide 1856 (EMBL, M80478), resulting in Phe55(TTT)-->Ser(TCT) replacement. This substitution created a new MnlI restriction site in the mutant allele. Restriction analysis revealed that the propositus was homozygous for this sequence, and the same mutation was not detected in 57 unrelated Japanese subjects. Since this mutation is located in the leucine-rich motif (LRM) of the GPIX polypeptide, the Phe55-->Ser substitution may result in an alteration of the LRM which leads to the impaired surface expression of GPIb/IX/V complex, a characteristic of BSS.

Auxotrophy of Pseudomonas aeruginosa in cystic fibrosis.

Seventy-four of 403 (18.4%) sputum isolates of Pseudomonas aeruginosa from 49 of 136 (36.0%) adults with cystic fibrosis (CF) were auxotrophic mutants. Two of 11 (18.2%) isolates of P. aeruginosa taken from patients with non-CF bronchiectasis were also auxotrophic. All 99 strains taken from non-bronchiectatic sources were prototrophic. Forty-six of 55 (83.6%) CF auxotrophs required one or more of 36 growth factors tested; the requirements for the remaining 9 isolates were not identified. Methionine was the sole factor required by 17 of 22 (77.3%) isolated which depended on a single factor. We conclude that auxotrophy is a feature of P. aeruginosa infection in cystic fibrosis.