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Jeffrey A. Moscow - One of the best experts on this subject based on the ideXlab platform.
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Revised version 03/01/2012 Title Page
2016Co-Authors: Jeffrey A. MoscowAbstract:Pharmacologic properties of polyethylene glycol-modified Bacillus thiaminolyticus Thiaminase I enzyme
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Metabolic Effects of Acute Thiamine Depletion Are Reversed by Rapamycin in Breast and Leukemia Cells
2016Co-Authors: Shuqian Liu, Sumitra Miriyala, Mignon A. Keaton, Craig T. Jordan, Christina Wiedl, Daret K. St. Clair, Jeffrey A. MoscowAbstract:Thiamine-dependent enzymes (TDEs) control metabolic pathways that are frequently altered in cancer and therefore present cancer-relevant targets. We have previously shown that the recombinant enzyme Thiaminase cleaves and depletes intracellular thiamine, has growth inhibitory activity against leukemia and breast cancer cell lines, and that its growth inhibitory effects were reversed in leukemia cell lines by rapamycin. Now, we first show further evidence of Thiaminase therapeutic potential by demonstrating its activity against breast and leukemia xenografts, and against a primary leukemia xenograft. We therefore further explored the metabolic effects of Thiaminase in combination with rapamycin in leukemia and breast cell lines. Thiaminase decreased oxygen consumption rate and increased extracellular acidification rate, consistent with the inhibitory effect of acute thiamine depletion on the activity of the TDEs pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes; these effects were reversed by rapamycin. Metabolomic studies demonstrated intracellular thiamine depletion and the presence of the thiazole cleavage product in Thiaminase-treated cells, providing validation of the experimental procedures. Accumulation of ribose and ribulose in both cell lines support the Thiaminase-mediated suppression of the TDE transketolase. Interestingly, Thiaminase suppression of another TDE, branched chain amino ketoacid dehydrogenase (BCKDH), showed very different patterns in the two cell lines: in RS4 leukemia cells it led to an increase in BCKDH substrates, and in MCF-7 breast cancer cells it led to a decrease in BCKDH products. Immunoblo
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Effect of thiamine antagonists on Thiaminase activity.
2014Co-Authors: Shuqian Liu, Sumitra Miriyala, Mignon A. Keaton, Craig T. Jordan, Christina Wiedl, Daret K. St. Clair, Jeffrey A. MoscowAbstract:A. Growth inhibition of Reh and RS4 leukemia cells in the presence or absence of Thiaminase and either N3PT or oxythiamine. B. Growth inhibition of RS4 and Reh leukemia cells incubated in normal medium, thiamine-free medium or medium containing Thiaminase prior to exposure to different concentrations of N3PT. C. RS4 subcutaneous xenografts showing untreated control, N3PT alone, 1k-PEGylated Thiaminase and 1k-PEGylated Thiaminase followed by N3PT. The time-to-endpoint was 16.5 days for control, 23 days for N3PT, 25 days for 1k-PEG Thiaminase and 55 days for 1k-PEG Thiaminase (p
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Validation of Thiaminase action in of RS4 leukemia cells and MCF-7 breast cancer cells by metabolomic analysis.
2014Co-Authors: Shuqian Liu, Sumitra Miriyala, Mignon A. Keaton, Craig T. Jordan, Christina Wiedl, Daret K. St. Clair, Jeffrey A. MoscowAbstract:Both cell lines were analyzed under six conditions: control for 24 hours (C-24); incubation in Thiaminase for 24 hours (T-24); control for 48 hours (C-48); Thiaminase for 48 hours (T-48); rapamycin for 48 hours (R-48); and both rapamycin and Thiaminase for 48 hours (R+T-48). The median is indicated by the bar in the center of the rectangle, the rectangle dimensions reflect the range of the two mid-quartile values, and the outer bars represent the ranges of all of the values. The data represent four independent experiments. For C-48 vs T-48 and C-48 vs T+R-48 comparisons, ** indicates p
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Differential effects of Thiaminase on branched chain amino acid catabolism.
2014Co-Authors: Shuqian Liu, Sumitra Miriyala, Mignon A. Keaton, Craig T. Jordan, Christina Wiedl, Daret K. St. Clair, Jeffrey A. MoscowAbstract:A. Schematic diagram showing branched chain amino acid catabolism. B. Different metabolomic signatures indicating inhibition of BCKDH by Thiaminase. The top three panels show accumulation of BCKDH substrates after 48-7 cells after Thiaminase treatment- most notably isovalerylcarnitine, which is also reversed by rapamycin. C. An immunoblot of cytosolic and mitochondrial branched chain amino acid transferase (cBCAT and mBCAT), the enzymes that catalyzes the reactions that produce BCKDH substrates, and total and phosphorylated BCKDH subunit E1 (BCKD-E1 and pBCKD-E1, respectively) in RS4 leukemia cells treated with Thiaminase, rapamycin or both. For C-48 vs T-48 and C-48 vs T+R-48 comparisons, ** indicates p
Tadhg P. Begley - One of the best experts on this subject based on the ideXlab platform.
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structure of a clostridium botulinum c143s Thiaminase i thiamin complex reveals active site architecture
Biochemistry, 2013Co-Authors: Megan D Sikowitz, Tadhg P. Begley, Brateen Shome, Yang Zhang, Steven E EalickAbstract:Thiaminases are responsible for the degradation of thiamin and its metabolites. Two classes of Thiaminases have been identified based on their three-dimensional structures and their requirements for a nucleophilic second substrate. Although the reactions of several Thiaminases have been characterized, the physiological role of thiamin degradation is not fully understood. We have determined the three-dimensional X-ray structure of an inactive C143S mutant of Clostridium botulinum (Cb) Thiaminase I with bound thiamin at 2.2 A resolution. The C143S/thiamin complex provides atomic level details of the orientation of thiamin upon binding to Cb-Thiaminase I and the identity of active site residues involved in substrate binding and catalysis. The specific roles of active site residues were probed by using site directed mutagenesis and kinetic analyses, leading to a detailed mechanism for Cb-Thiaminase I. The structure of Cb-Thiaminase I is also compared to the functionally similar but structurally distinct Thiaminase II.
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thiamine deficiency in cambodian infants with and without beriberi
The Journal of Pediatrics, 2012Co-Authors: Debra Coats, Kelsey Sheltondodge, Kevanna Ou, Vannara Khun, Sommon Seab, Chiva Prou, Silvia Tortorelli, Lisa E Cooper, Thomas P Moyer, Tadhg P. BegleyAbstract:Objectives To test the hypothesis that heavy metal toxicity and consumption of Thiaminase-containing foods predispose to symptomatic thiamine deficiency. Study design In a case-control study, thiamine diphosphate (TDP) blood concentrations were measured in 27 infants diagnosed with beriberi at a rural clinic, as well as their mothers and healthy Cambodian and American controls. Blood and urine levels of lead, arsenic, cadmium, mercury, and thallium were measured. Local food samples were analyzed for Thiaminase activity. Results Mean TDP level among cases and Cambodian controls was 48 and 56 nmol/L, respectively ( P = .08) and was 132 nmol/L in American controls ( P P = .92), and was 126 nmol/L in American mothers ( P P = .02). Infant TDP level decreased with infant age and was positively associated with maternal TDP level. Specific diagnostic criteria for beriberi did not correlate with TDP level. There was no correlation between heavy metal levels and either TDP level or case/control status. No Thiaminase activity was observed in food samples. Conclusions Thiamine deficiency is endemic among infants and nursing mothers in rural southeastern Cambodia and is often clinically inapparent. Neither heavy metal toxicity nor consumption of Thiaminase-containing foods account for thiamine deficiency in this region.
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comparison of Thiaminase activity in fish using the radiometric and 4 nitrothiophenol colorimetric methods
Journal of Great Lakes Research, 2010Co-Authors: Dale C Honeyfield, Lisa R Brown, Clifford E Kraft, Jeremiah Hanes, Tadhg P. BegleyAbstract:Thiaminase induced thiamine deficiency occurs in fish, humans, livestock and wild animals. A non-radioactive Thiaminase assay was described in 2007, but a direct comparison with the radioactive 14C-thiamine method which has been in use for more than 30 years has not been reported. The objective was to measure Thiaminase activity in forage fish (alewife Alosa pseudoharengus, rainbow smelt Osmerus mordax, and slimy sculpin Cottus cognatus) consumed by predators that manifest thiamine deficiency using both methods. Modifications were made to the colorimetric assay to improve repeatability. Modification included a change in assay pH, enhanced sample clean-up, constant assay temperature (37 °C), increase in the concentration of 4-nitrothiophenol (4NTP) and use of a spectrophotometer fitted with a 0.2 cm cell. A strong relationship between the two assays was found for 51 alewife (R2 = 0.85), 36 smelt (R2 = 0.87) and 20 sculpin (R2 = 0.82). Thiaminase activity in the colorimetric assay was about 1000 times higher than activity measured by the radioactive method. Application of the assay to fish species from which no Thiaminase activity has previously been reported resulted in no 4NTP Thiaminase activity being found in bloater Coregonus hoyi, lake trout Salvelinus namaycusch, steelhead trout Oncorhynchus mykiss or Chinook salmon Oncorhynchus tshawytscha. In species previously reported to contain Thiaminase, 4NTP Thiaminase activity was measured in bacteria Paenibacillus thiaminolyticus, gizzard shad Dorosoma cepedianum, bracken fern Pteridium aquilinum, quagga mussel Dreissena bugensis and zebra mussels D. polymorpha.
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sensitivity of breast cancer cell lines to recombinant Thiaminase i
Cancer Chemotherapy and Pharmacology, 2010Co-Authors: Shuqian Liu, Tadhg P. Begley, Noel R Monks, Jeremiah Hanes, Jeffrey A. MoscowAbstract:We have previously shown that the expression of the thiamine transporter THTR2 is decreased sevenfold in breast cancer, which may leave breast cancer cells vulnerable to acute thiamine starvation. This concept was supported by the observation that MDA231 breast cancer xenografts demonstrated growth inhibition in mice fed a thiamine-free diet. We purified recombinant Bacillus thiaminolyticus Thiaminase I enzyme, which digests thiamine, to study acute thiamine starvation in breast cancer. Thiaminase I enzyme was cytotoxic in six breast cancer cell lines with IC50s ranging from 0.012 to 0.022 U/ml. The growth inhibitory effects of the combination of Thiaminase I with either doxorubicin or paclitaxel were also examined. Over a wide range of drug concentrations, Thiaminase 1 was consistently synergistic or additive with doxorubicin and paclitaxel in MCF-7, ZR75, HS578T and T47D cell lines, with most combinations having a calculated combination index (CI) of less than 0.8, indicating synergy. Although Thiaminase I exposure did not stimulate the energy-sensing signaling kinases AKT, AMPK and GSK-3β in MCF-7, ZR75, HS578T and T47D cell lines, Thiaminase I exposure did stimulate expression of the ER stress response protein GRP78. In summary, Thiaminase I is cytotoxic in breast cancer cell lines and triggers the unfolded protein response. These findings suggest that THTR2 down-regulation in breast tumors may present a nutritional vulnerability that could be exploited by Thiaminase I enzyme therapy.
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an assay for Thiaminase i in complex biological samples
Analytical Biochemistry, 2007Co-Authors: Jeremiah Hanes, Clifford E Kraft, Tadhg P. BegleyAbstract:An alternative method for measuring Thiaminase I activity in complex samples is described. This assay is based on the selective consumption of the highly chromophoric 4-nitrothiophenolate by Thiaminase I, resulting in a large decrease in absorbance at 411 nm. This new assay is simple and sensitive, and it requires only readily available chemicals and a visible region spectrophotometer. In addition, the assay is optimized for high-throughput analysis in a 96-well format with complex biological samples. Published by Elsevier Inc.
Shuqian Liu - One of the best experts on this subject based on the ideXlab platform.
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Metabolic Effects of Acute Thiamine Depletion Are Reversed by Rapamycin in Breast and Leukemia Cells
2016Co-Authors: Shuqian Liu, Sumitra Miriyala, Mignon A. Keaton, Craig T. Jordan, Christina Wiedl, Daret K. St. Clair, Jeffrey A. MoscowAbstract:Thiamine-dependent enzymes (TDEs) control metabolic pathways that are frequently altered in cancer and therefore present cancer-relevant targets. We have previously shown that the recombinant enzyme Thiaminase cleaves and depletes intracellular thiamine, has growth inhibitory activity against leukemia and breast cancer cell lines, and that its growth inhibitory effects were reversed in leukemia cell lines by rapamycin. Now, we first show further evidence of Thiaminase therapeutic potential by demonstrating its activity against breast and leukemia xenografts, and against a primary leukemia xenograft. We therefore further explored the metabolic effects of Thiaminase in combination with rapamycin in leukemia and breast cell lines. Thiaminase decreased oxygen consumption rate and increased extracellular acidification rate, consistent with the inhibitory effect of acute thiamine depletion on the activity of the TDEs pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes; these effects were reversed by rapamycin. Metabolomic studies demonstrated intracellular thiamine depletion and the presence of the thiazole cleavage product in Thiaminase-treated cells, providing validation of the experimental procedures. Accumulation of ribose and ribulose in both cell lines support the Thiaminase-mediated suppression of the TDE transketolase. Interestingly, Thiaminase suppression of another TDE, branched chain amino ketoacid dehydrogenase (BCKDH), showed very different patterns in the two cell lines: in RS4 leukemia cells it led to an increase in BCKDH substrates, and in MCF-7 breast cancer cells it led to a decrease in BCKDH products. Immunoblo
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Effect of thiamine antagonists on Thiaminase activity.
2014Co-Authors: Shuqian Liu, Sumitra Miriyala, Mignon A. Keaton, Craig T. Jordan, Christina Wiedl, Daret K. St. Clair, Jeffrey A. MoscowAbstract:A. Growth inhibition of Reh and RS4 leukemia cells in the presence or absence of Thiaminase and either N3PT or oxythiamine. B. Growth inhibition of RS4 and Reh leukemia cells incubated in normal medium, thiamine-free medium or medium containing Thiaminase prior to exposure to different concentrations of N3PT. C. RS4 subcutaneous xenografts showing untreated control, N3PT alone, 1k-PEGylated Thiaminase and 1k-PEGylated Thiaminase followed by N3PT. The time-to-endpoint was 16.5 days for control, 23 days for N3PT, 25 days for 1k-PEG Thiaminase and 55 days for 1k-PEG Thiaminase (p
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Validation of Thiaminase action in of RS4 leukemia cells and MCF-7 breast cancer cells by metabolomic analysis.
2014Co-Authors: Shuqian Liu, Sumitra Miriyala, Mignon A. Keaton, Craig T. Jordan, Christina Wiedl, Daret K. St. Clair, Jeffrey A. MoscowAbstract:Both cell lines were analyzed under six conditions: control for 24 hours (C-24); incubation in Thiaminase for 24 hours (T-24); control for 48 hours (C-48); Thiaminase for 48 hours (T-48); rapamycin for 48 hours (R-48); and both rapamycin and Thiaminase for 48 hours (R+T-48). The median is indicated by the bar in the center of the rectangle, the rectangle dimensions reflect the range of the two mid-quartile values, and the outer bars represent the ranges of all of the values. The data represent four independent experiments. For C-48 vs T-48 and C-48 vs T+R-48 comparisons, ** indicates p
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Differential effects of Thiaminase on branched chain amino acid catabolism.
2014Co-Authors: Shuqian Liu, Sumitra Miriyala, Mignon A. Keaton, Craig T. Jordan, Christina Wiedl, Daret K. St. Clair, Jeffrey A. MoscowAbstract:A. Schematic diagram showing branched chain amino acid catabolism. B. Different metabolomic signatures indicating inhibition of BCKDH by Thiaminase. The top three panels show accumulation of BCKDH substrates after 48-7 cells after Thiaminase treatment- most notably isovalerylcarnitine, which is also reversed by rapamycin. C. An immunoblot of cytosolic and mitochondrial branched chain amino acid transferase (cBCAT and mBCAT), the enzymes that catalyzes the reactions that produce BCKDH substrates, and total and phosphorylated BCKDH subunit E1 (BCKD-E1 and pBCKD-E1, respectively) in RS4 leukemia cells treated with Thiaminase, rapamycin or both. For C-48 vs T-48 and C-48 vs T+R-48 comparisons, ** indicates p
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Effects of Thiaminase on aromatic amino acid catabolism.
2014Co-Authors: Shuqian Liu, Sumitra Miriyala, Mignon A. Keaton, Craig T. Jordan, Christina Wiedl, Daret K. St. Clair, Jeffrey A. MoscowAbstract:A. Accumulation of the products of phenylalanine (phenylpyruvate and phenyllactate) and tyrosine (hydroxyphenylpyruvate and hydroxyphenyllactate) catabolism in RS4 cells after treatment with Thiaminase for 48 hours. B. Accumulation of tryptophan catabolites after Thiaminase treatment, showing accumulation of indolelactate but not kynurenine. For C-48 vs T-48 and C-48 vs T+R-48 comparisons, ** indicates p
Donald E Tillitt - One of the best experts on this subject based on the ideXlab platform.
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paenibacillus thiaminolyticus is not the cause of thiamine deficiency impeding lake trout salvelinus namaycush recruitment in the great lakes
Canadian Journal of Fisheries and Aquatic Sciences, 2012Co-Authors: Catherine A Richter, James L Zajicek, Allison N Evans, Maureen K Wrightosment, Scott A Heppell, Stephen C Riley, Charles C Krueger, Donald E TillittAbstract:Thiamine (vitamin B1) deficiency is a global concern affecting wildlife, livestock, and humans. In Great Lakes salmonines, thiamine deficiency causes embryo mortality and is an impediment to restoration of native lake trout (Salvelinus namaycush) stocks. Thiamine deficiency in fish may result from a diet of prey with high levels of Thiaminase I. The discov- eries that the bacterial species Paenibacillus thiaminolyticus produces Thiaminase I, is found in viscera of Thiaminase- containing prey fish, and causes mortality when fed to lake trout in the laboratory provided circumstantial evidence implicat- ing P. thiaminolyticus. This study quantified the contribution of P. thiaminolyticus to the total Thiaminase I activity in multi- ple trophic levels of Great Lakes food webs. Unexpectedly, no relationship between Thiaminase activity and either the amount of P. thiaminolyticus Thiaminase I protein or the abundance of P. thiaminolyticus cells was found. These results dem- onstrate that P. thiaminolyticus is not the primary source of Thiaminase activity affecting Great Lakes salmonines and calls into question the long-standing assumption that P. thiaminolyticus is the source of Thiaminase in other wild and domestic animals. Resume : La carence en thiamine (vitamine B1) est un probleme d'envergure planetaire touchant tant les animaux sauvages et d'elevage que les humains. Chez les salmonines des Grands Lacs, la carence en thiamine est cause de mortalite embryon- naire et entrave le retablissement des stocks indigenes de touladi (Salvelinus namaycush). Une carence en thiamine chez les poissons peut decouler d'une alimentation faite des proies avec de fortes teneurs en Thiaminase I. Le fait que la bacterie Pae- nibacillus thiaminolyticus produit de la Thiaminase I, qu'elle est presente dans les visceres de poissons proies contenant de la Thiaminase et qu'elle cause une mortalite quand elle est donnee a manger a des touladis en laboratoire sont autant de de- couvertes offrant des preuves circonstancielles de l'implication de P. thiaminolyticus. L'etude a quantifie la contribution de P. thiaminolyticus al 'activite totale de la Thiaminase I dans de multiples niveaux trophiques de reseaux trophiques des
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variation in lake michigan alewife alosa pseudoharengus Thiaminase and fatty acids composition
Journal of Freshwater Ecology, 2010Co-Authors: Dale C Honeyfield, John D Fitzsimons, Donald E Tillitt, Scott B BrownAbstract:ABSTRACT Thiaminase activity of alewife (Alosa pseudoharengus) is variable across Lake Michigan, yet factors that contribute to the variability in alewife Thiaminase activity are unknown. The fatty acid content of Lake Michigan alewife has not been previously reported. Analysis of 53 Lake Michigan alewives found a positive correlation between Thiaminase activity and the following fatty acid C22:1n9, sum of omega-6 fatty acids (SW6), and sum of the polyunsaturated fatty acids. Thiaminase activity was negatively correlated with C15:0, C16:0, C17:0, C18:0, C20:0, C22:0, C24:0, C18:1n9t, C22:2, and the sum of all saturated fatty acids (SAFA). Multi-variant regression analysis resulted in three variables (C18:1n9t, Sw6, SAFA) that explained 71% (R2 =0.71, P<0.0001) of the variation in Thiaminase activity. Because the fatty acid content of an organism is related is food source, diet may be an important factor modulating alewife Thiaminase activity. These data suggest there is an association between fatty acids ...
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variations of Thiaminase i activity ph dependencies among typical great lakes forage fish and paenibacillus thiaminolyticus
Journal of Aquatic Animal Health, 2009Co-Authors: James L Zajicek, Scott B Brown, Lisa R Brown, John D Fitzsimons, Dale C Honeyfield, Donald E TillittAbstract:Abstract The source of Thiaminase in the Great Lakes food web remains unknown. Biochemical characterization of the Thiaminase I activities observed in forage fish was undertaken to provide insights into potential Thiaminase sources and to optimize catalytic assay conditions. We measured the Thiaminase I activities of crude extracts from five forage fish species and one strain of Paenibacillus thiaminolyticus over a range of pH values. The clupeids, alewife Alosa pseudoharengus and gizzard shad Dorosoma cepedianum, had very similar Thiaminase I pH dependencies, with optimal activity ranges (≥90% of maximum activity) between pH 4.6 and 5.5. Rainbow smelt Osmerus mordax and spottail shiner Notropis hudsonius had optimal activity ranges between pH 5.5–6.6. The Thiaminase I activity pH dependence profile of P. thiaminolyticus had an optimal activity range between pH 5.4 and 6.3, which was similar to the optimal range for rainbow smelt and spottail shiners. Incubation of P. thiaminolyticus extracts with extract...
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quantitative polymerase chain reaction pcr assays for a bacterial Thiaminase i gene and the Thiaminase producing bacterium paenibacillus thiaminolyticus
Journal of Aquatic Animal Health, 2009Co-Authors: Catherine A Richter, Dale C Honeyfield, James L Zajicek, Maureen K Wrightosment, Donald E TillittAbstract:Abstract The Thiaminase I enzyme produced by the gram-positive bacterium Paenibacillus thiaminolyticus isolated from the viscera of Lake Michigan alewives Alosa pseudoharengus is currently the only defined source of the Thiaminase activity linked to thiamine (vitamin B1) deficiency in early mortality syndrome (EMS) in the larvae of Great Lakes salmonines. Diets of alewife or isolated strains of P. thiaminolyticus mixed in a semipurified diet and fed to lake trout Salvelinus namaycush have been shown to produce EMS in fry. We utilized quantitative polymerase chain reaction (Q-PCR) to aid in studies of the sources of P. thiaminolyticus and Thiaminase I. Quantitative PCR assays were established to detect the Thiaminase I gene of P. thiaminolyticus, the 16S rRNA gene from most species of bacteria, and the 16S rRNA gene specifically from P. thiaminolyticus and a few closely related taxa. The Q-PCR assays are linear over at least six orders of magnitude and can detect the Thiaminase I gene of P. thiaminolyticus...
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dreissenid mussels from the great lakes contain elevated Thiaminase activity
Journal of Great Lakes Research, 2009Co-Authors: Donald E Tillitt, James L Zajicek, Catherine A Richter, Allison N Evans, Stephen C Riley, Jerrine S Nichols, Jacques Rinchard, Charles C KruegerAbstract:ABSTRACT We examined Thiaminase activity in dreissenid mussels collected at different depths and seasons, and from various locations in Lakes Michigan, Ontario, and Huron. Here we present evidence that two dreissenid mussel species (Dreissena bugensis and D. polymorpha) contain Thiaminase activity that is 5–100 fold greater than observed in Great Lakes fishes. Thiaminase activity in zebra mussels ranged from 10,600 to 47,900 pmol g-1·min-1 and activities in quagga mussels ranged from 19,500 to 223,800 pmol g-1 ·min-1. Activity in the mussels was greatest in spring, less in summer, and least in fall. Additionally, we observed greater Thiaminase activity in dreissenid mussels collected at shallow depths compared to mussels collected at deeper depths. Dreissenids constitute a significant and previously unknown pool of Thiaminase in the Great Lakes food web compared to other known sources of this thiamine (vitamin B1)-degrading enzyme. Thiaminase in forage fish of the Great Lakes has been causally linked to t...
Esther R Angert - One of the best experts on this subject based on the ideXlab platform.
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Thiaminase i provides a growth advantage by salvaging precursors from environmental thiamine and its analogs in burkholderia thailandensis
Applied and Environmental Microbiology, 2018Co-Authors: David R Sannino, Clifford E Kraft, Katie A Edwards, Esther R AngertAbstract:ABSTRACT Thiamine is essential to life, as it serves as a cofactor for enzymes involved in critical carbon transformations. Many bacteria can synthesize thiamine, while thiamine auxotrophs must obtain it or its precursors from the environment. Thiaminases degrade thiamine by catalyzing the base-exchange substitution of thiazole with a nucleophile, and Thiaminase I specifically has been implicated in thiamine deficiency syndromes in animals. The biological role of this secreted enzyme has been a long-standing mystery. We used the Thiaminase I-producing soil bacterium Burkholderia thailandensis as a model to ascertain its function. First, we generated thiamine auxotrophs, which are still able to use exogenous precursors (thiazole and hydroxymethyl pyrimidine), to synthesize thiamine. We found that Thiaminase I extended the survival of these strains, when grown in defined media where thiamine was serially diluted out, compared to isogenic strains that could not produce Thiaminase I. Thiamine auxotrophs grew better on thiamine precursors than thiamine itself, suggesting Thiaminase I functions to convert thiamine to useful precursors. Furthermore, our findings demonstrate that Thiaminase I cleaves phosphorylated thiamine and toxic analogs, which releases precursors that can then be used for thiamine synthesis. This study establishes a biological role for this perplexing enzyme and provides additional insight into the complicated nature of thiamine metabolism and how individual bacteria may manipulate the availability of a vital nutrient in the environment. IMPORTANCE The function of Thiaminase I has remained a long-standing, unsolved mystery. The enzyme is only known to be produced by a small subset of microorganisms, although Thiaminase I activity has been associated with numerous plants and animals, and is implicated in thiamine deficiencies brought on by consumption of organisms containing this enzyme. Genomic and biochemical analyses have shed light on potential roles for the enzyme. Using the genetically amenable Thiaminase I-producing soil bacterium Burkholderia thailandensis, we were able to demonstrate that Thiaminase I helps salvage precursors from thiamine derivatives in the environment and degrades thiamine to its precursors, which are preferentially used by B. thailandensis auxotrophs. Our study establishes a biological role for this perplexing enzyme and provides insight into the complicated nature of thiamine metabolism. It also establishes B. thailandensis as a robust model system for studying thiamine metabolism.
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Genomic insights into the thiamin metabolism of Paenibacillus thiaminolyticus NRRL B-4156 and P. apiarius NRRL B-23460
'Springer Science and Business Media LLC', 2017Co-Authors: David Sannino, Esther R AngertAbstract:Abstract Paenibacillus thiaminolyticus is the model organism for studying Thiaminase I, an enigmatic extracellular enzyme. Originally isolated from the feces of clinical patients suffering from thiamin deficiency, P. thiaminolyticus has been implicated in thiamin deficiencies in humans and other animals due to its ability to produce this thiamin-degrading enzyme. Its close relative, P. apiarius, also produces Thiaminase I and was originally isolated from dead honeybee larvae, though it has not been reported to be a honeybee pathogen. We generated draft genomes of the type strains of both species, P. thiaminolyticus NRRL B-4156 and P. apiarius NRRL B-23460, to deeply explore potential routes of thiamin metabolism. We discovered that the Thiaminase I gene is located in a highly conserved operon with thiamin biosynthesis and salvage genes, as well as genes involved in the biosynthesis of the antibiotic bacimethrin. Based on metabolic pathway predictions, P. apiarius NRRL B-23460 has the genomic capacity to synthesize thiamin de novo using a pathway that is rarely seen in bacteria, but P. thiaminolyticus NRRL B-4156 is a thiamin auxotroph. Both genomes encode importers for thiamin and the pyrimidine moiety of thiamin, as well as enzymes to synthesize thiamin from pyrimidine and thiazole
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a rapid method for assaying Thiaminase i activity in diverse biological samples
PLOS ONE, 2014Co-Authors: Clifford E Kraft, Eric R. L. Gordon, Esther R AngertAbstract:Vitamin B1 (thiamine) deficiencies can lead to neurological disorders, reproductive failure and death in wild and domestic animal populations. In some cases, disease is brought about by the consumption of foods high in Thiaminase I activity. Levels of Thiaminase activity in these foods are highly variable and the factors leading to production of this enzyme are poorly understood. Here we describe improvements in a spectrophotometric Thiaminase I activity assay that measures the disappearance of 4-nitrothiophenol, a favored nucleophile co-substrate that replaces the thiazole portion of thiamine during the inactivation of thiamine by the enzyme. Scalable sample processing protocols and a 96-well microtiter plate format are presented that allow the rapid evaluation of multiple, replicated samples in the course of only a few hours. Observed levels of activity in bacterial culture supernatant, fish, ferns and molluscs using this colorimetric assay were similar to previously published reports that employed a radiometric method. Organisms devoid of Thiaminase I, based upon previous work, showed no activity with this assay. In addition, activity was found in a variety of fishes and one fern species from which this enzyme had not previously been reported. Overall, we demonstrate the suitability of this technique for measuring Thiaminase I activity within small amounts of tissue and environmental samples with replication levels that were heretofore prohibitive. The assay provides a considerable improvement in the ability to examine and understand the properties of an enzyme that has a substantial influence on organism and ecosystem health.
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Thiaminase I activity in New York woodland ferns.
2014Co-Authors: Clifford E Kraft, Eric R. L. Gordon, Esther R AngertAbstract:Replicate samples (number of replicates shown above the plotted activity value) from rhizomes or fine roots of ferns were assayed. Bracken fern Thiaminase I activity was similar to previously published reports. Thiaminase I activity was detected in only one additional fern species (Christmas fern). Error bars represent the standard error of replicates.
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4-NTP assay detection of Thiaminase I activity in bacteria known to produce this thiamine-degrading enzyme.
2014Co-Authors: Clifford E Kraft, Eric R. L. Gordon, Esther R AngertAbstract:Cell-free supernatant from replicate 1-day old cultures of C. sporogenes and P. thiaminolyticus were tested for Thiaminase activity using the 4-NTP assay. Error bars represent the standard error of three replicates.