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Information on EC 2.5.1.2 - thiamine pyridinylase and Organism(s) Paenibacillus thiaminolyticus and UniProt Accession P45741
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2.5.1.2 thiamine pyridinylaseIUBMB Comments
Various bases and thiol compounds can act instead of pyridine.
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Word Map
- 2.5.1.2
- thiaminolyticus
- medicine
- thiazole
- aniline
- pseudoharengus
- viscera
-
bivalve
-
alosa
- analysis
- nutrition
The taxonomic range for the selected organisms is: Paenibacillus thiaminolyticus
The expected taxonomic range for this enzyme is: Eukaryota, Bacteria
The expected taxonomic range for this enzyme is: Eukaryota, Bacteria
Reaction Schemes
Synonyms
thiaminase i, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
pyrimidine transferase
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-
-
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thiamin hydrolase
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-
-
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thiamin pyridinolase
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thiamin pyridinylase
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-
-
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thiaminase
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-
-
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thiaminase I
thiamine pyridinolase
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thiamine pyridinylase
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thiaminase I
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-
-
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pyrimidyl group transfer
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-
-
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PATHWAY SOURCE
PATHWAYS
KEGG
SYSTEMATIC NAME
IUBMB Comments
thiamine:base 2-methyl-4-aminopyrimidine-5-methenyltransferase
Various bases and thiol compounds can act instead of pyridine.
CAS REGISTRY NUMBER
COMMENTARY
9030-35-7
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SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
thiamine
?
there is degradation of thiamine without a base substrate, possibly the ring-opened form of thiamine functions as the nucleophile
-
-
?
thiamine + 4-nitrothiophenolate
4-methyl-5-(2-hydroxyethyl)thiazole + 2-methyl-5[[(4-nitrophenyl)sulfanyl]methyl]pyrimidin-4-amine
no activity with thiaminase II
-
-
?
thiamine + veratrylamine
bis-(4-amino-2-methyl)pyrimidinyl-3,4-dimethoxybenzylamine + 4-methyl-5-(2-hydroxyethyl)-thiazole
the reaction can occur without base substrate
-
?
thiamine + aniline
5-(anilinomethyl)-2-methylpyrimidin-4-amine + 4-methyl-5-(2-hydroxyethyl)-thiazole
thiamine + quinoline
4-methyl-5-(2-hydroxyethyl)-thiazole + 1-[(4-amino-2-methylpyrimidin-5-yl)methyl]quinolinium
-
-
-
-
?
additional information
?
-
-
primary substrates inactivate the enzyme at 0.006 mM and secondary substrates or acceptor bases reactivate the enzyme at 10 mM, each substrate at high concentrations eliminates the inactivation of the other substrate
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-
?
thiamine + aniline
5-(anilinomethyl)-2-methylpyrimidin-4-amine + 4-methyl-5-(2-hydroxyethyl)-thiazole
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-
-
?
thiamine + aniline
5-(anilinomethyl)-2-methylpyrimidin-4-amine + 4-methyl-5-(2-hydroxyethyl)-thiazole
-
-
-
?
thiamine + aniline
5-(anilinomethyl)-2-methylpyrimidin-4-amine + 4-methyl-5-(2-hydroxyethyl)-thiazole
-
-
-
?
thiamine + aniline
5-(anilinomethyl)-2-methylpyrimidin-4-amine + 4-methyl-5-(2-hydroxyethyl)-thiazole
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-
-
?
thiamine + aniline
5-(anilinomethyl)-2-methylpyrimidin-4-amine + 4-methyl-5-(2-hydroxyethyl)-thiazole
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one molecule of thiamine is cleaved releasing one molecule of methylhydroxyethylthiazole for one molecule of enzyme
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?
thiamine + pyridine
heteropyrithiamine + 4-methyl-5-(2-hydroxyethyl)-thiazole
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-
-
?
thiamine + pyridine
heteropyrithiamine + 4-methyl-5-(2-hydroxyethyl)-thiazole
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-
-
?
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
4-Amino-5-(anilinomethyl)-6-chloro-2-methylpyrimidine
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complete irreversible inhibition 2 times faster than 4-amino-6-chloro-2-methylpyrimidine
4-Amino-6-chloro-2-methylpyrimidine
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complete irreversible inhibition, activity protected by thiamine and quinolone
p-chloromercuribenzoate
-
50% of inhibition at 0.2 mM and inhibits reactivation by pyridine
4-Amino-6-chloro-2-methylpyrimidine
this suicide substrate is linked to Cys113 of the enzyme
Heteropyrithiamine
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inactivation at 0.006 mM that is restored by pyridine at 25 mM and inhibition of restoration of activity by pyridine at 1 mM
thiamine
-
inactivation is proportional to thiamine concentration and the thiamine/enzyme ratio is 1 mol/1 mol
additional information
-
chloramphenicol does not affect activity suggesting that the enzyme has an inactive form
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additional information
-
primary substrates inactivate the enzyme at 0.006 mM, the lost activity can be restored by incubation at 37°C for 60 min after dialysis or by incubation with secondary substrates or acceptor bases, each substrate at high concentrations abolishes the inactivation of the other substrate
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additional information
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acidic conditions prevent inactivation but low pH does not restore activity after inactivation, quaternary nitrogen linked to a substituted pyrimidine ring through a methylene bridge is shared by compounds that inactivates the enzyme, inactivation alters either the net charge or the structure of the protein or both
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additional information
-
4-amino-2-methyl pyrimidine does not inactivate nor competitively inhibits the enzyme
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
aromatic amines
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increased activity, aniline is the most potent activator, primary amines not directly attached to the benzene ring have no effect
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Pyridine
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at 25 mM restores activity inhibited by heteropyrimidine at 0.006 mM
sulfhydryl compounds
-
increase activity, more markedly in bacteria
2-mercaptoethanol
-
similar effects as pyridine at 10 mM probably due to its action as secondary substrate
2-mercaptoethanol
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reactivates enzyme less effectively than dithiothreitol
dithiothreitol
-
similar effects as pyridine at 10 mM probably due to its action as secondary substrate
heterocyclic amines
-
pyridine, 2-amino-pyridine, 3-amino-pyridine increase activity
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heterocyclic amines
-
nicotinic acid and quinolone increase activity
-
heterocyclic amines
-
most 4-aminopyrimidines increase activity
-
additional information
-
aliphatic amines and taurine have no effect
-
additional information
-
L-phenylalanine, L-tyrosine, L-tryptophan and gamma-aceto-gammachloropropyl acetate have no effect
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.0364
4-nitrothiophenolate
pH 7.2, temperature not specified in the publication
2.9
aniline
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pH 5.8, 25°C, the enzyme does not follow strict Michaelis-Menten kinetics
0.0087
thiamine
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pH 5.8, 25°C, the enzyme does not follow strict Michaelis-Menten kinetics
0.0211
thiamine
pH 7.2, temperature not specified in the publication
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
297
4-nitrothiophenolate
pH 7.2, temperature not specified in the publication
260
thiamine
pH 7.2, temperature not specified in the publication
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
8380
4-nitrothiophenolate
pH 7.2, temperature not specified in the publication
12400
thiamine
pH 7.2, temperature not specified in the publication
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.096
4-Amino-5-(anilinomethyl)-6-chloro-2-methylpyrimidine
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
additional information
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a 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 assay is simple and sensitive, and it requires only readily available chemicals and a visible region spectrophotometer. The assay is optimized for high-throughput analysis in a 96-well format with complex biological samples
additional information
a 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 assay is simple and sensitive, and it requires only readily available chemicals and a visible region spectrophotometer. The assay is optimized for high-throughput analysis in a 96-well format with complex biological samples
additional information
-
method for measuring thiaminase activity in complex samples. The assay is based on the selective consumption of the highly chromophoric 4-nitrothiophenolate by thiaminase I, which is able to use a variety of nucleophiles as cosubstrates. This assay is sensitive and uses readily available chemicals and a visible region spectrophotometer. In addition, the assay can be easily performed in a high-throughput fashion in either 96- or 384-well plates
additional information
method for measuring thiaminase activity in complex samples. The assay is based on the selective consumption of the highly chromophoric 4-nitrothiophenolate by thiaminase I, which is able to use a variety of nucleophiles as cosubstrates. This assay is sensitive and uses readily available chemicals and a visible region spectrophotometer. In addition, the assay can be easily performed in a high-throughput fashion in either 96- or 384-well plates
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pH RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
37
37
-
after dialysis, 60 min of incubation restore 90% of activity inhibited by thiamine
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
cloned in Escherichia coli
SwissProt
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SOURCE
-
addition of yeast extract and Ca2+ in the medium increases production of thiaminase, addition of glutamate or brain heart infusion decreases production
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
physiological function
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although thiaminase I exposure does not stimulate the energy-sensing signaling kinases AKT, AMPK and GSK-3beta in MCF-7, ZR75, HS-578T and T-47D cell lines, thiaminase I exposure does stimulate expression of the ER stress response protein GRP78. Thiaminase I is cytotoxic in breast cancer cell lines and triggers the unfolded protein response
UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable).
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable). PDB
SCOP
CATH
UNIPROT
ORGANISM
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
a comparison of thiamin-binding protein tbpA from Escherichia coli and thiaminase-I, reveals structural similarity between the two proteins, specially in domain 1, suggesting that the two proteins evolve from a common ancestor
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
monomer
monomer
1 * 42000, SDS-polyacrylamide gel electrophoresis
monomer
active site between Pro109 and Phe118, includes nucleophilic site Cys113
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
7
50 mM sodium phosphate buffer, 2 mM dithiothreitol, 2 mM EDTA
636853, 636858
7
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50 mM sodium phosphate buffer, 2 mM dithiothreitol, 2 mM EDTA
636858
OXIDATION STABILITY
ORGANISM
UNIPROT
LITERATURE
addition of dithiothreitol, 2 mM, reduces the loss of activity
636853
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
by ammonium precipitation, DEAE column, Cibacron blue dye affinity column
by ammonium precipitation, Sephadex G-25 column, Sephadex G-100 column and DEAE-Sephadex column
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CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
cloned in Escherichia coli with Gly6 changed to Ala6 with no apparent effect
APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
analysis
-
improvements in a spectrophotometric thiaminase I activity assay that measures the disappearance of 4-nitrothiophenol providing scalable sample processing protocols and a 96-well microtiter plate format. Organisms devoid of thiaminase I, based upon previous work, show no activity with this assay. In addition, activity is found in a variety of fishes and one fern species from which this enzyme has not previously been reported
medicine
nutrition
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thiaminase can be used as an effective method for thiamine determination in food and fodder
medicine
-
bacteria can infect organisms and produce thiaminase disease
medicine
-
thiamine transporter THTR2 downregulation in breast tumors may present a nutritional vulnerability that can be exploited by thiaminase I enzyme therapy
medicine
-
modification of thiaminase with linear chain methox polyethylene glycol of 5 kDa eliminates cytotoxic activity in all cell lines tested. Both native thiaminase and 5k-PEGylated thiaminase efficiently depletes thiamine from cell culture medium, and both can use intracellular phosphorylated thiamine as substrates. Native enzyme more effectively depletes thiamine and thiamine diphosphate in RS4 leukemia cell cytosol, and native thiaminase depresses cellular respiration, whereas PEGylated thiaminase does not. Despite the lack of in vitro cytotoxicity, PEGylation markedly increases the in vivo toxicity of the enzyme. The half-life of native thiaminase is 1.5 h compared with 34.4 h for the 5k-PEGylated enzyme. Serum thiamine levels are depleted by both native and 5k-PEGylated enzyme. Despite superior pharmacokinetics, 5k-PEGylated thiaminase shows no antitumor effect against an RS4 leukemia xenograft, in contrast to native thiaminase
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Fujita, A.
Thiaminase
Adv. Enzymol. Relat. Subj. Biochem.
15
389-421
1954
Acanthogobius flavimanus, Anadara inflata, Aneurinibacillus aneurinilyticus, Aneurinibacillus aneurinilyticus BKA, Anguilla japonica, Athyrium nipponicum, Auxis hira, Carassius auratus, Celosia crista, Charybdis 6-dentata, Circe scripta, Clava kochi, Corbicula leana, Cristaria plicata, Cyprinus carpio, Dicranopteris linearis, Dicranopteris sp., Dryopteris erythrosora, Dryopteris lacera, Eleotris oxycephala, Equisetum arvense, Eviota abax, Gnathopogon mayedae, Haliotis gigantea, Haliotis japonica, Hypomesus olidus, Ischikauia steenackeri, Leptochilus ellipticus, Leucopsarion petersii, Lycopodium clavatum, Mactra quadrangularis, Mactra sulcataria, Meretrix meretrix, Misgurnus anguillicaudatus, Mugil cephalus, no activity in Astroconger myriaster, no activity in Cambaroides japonicus, no activity in Camposcia retusa, no activity in Metapenaeopsis acclivis, no activity in Plecoglossus altivelis, no activity in Squilla oratoria, Odontobutis obscura, Osmunda japonica, Ostrea laperousei, Paenibacillus thiaminolyticus, Paenibacillus thiaminolyticus BMM, Panulirus japonicus, Paphia philippinarum, Polystichum aculeatum, Polystichum falcatum, Polystichum fortunei, Polystichum tsus-simense, Portunus trituberculatus, Pteridium aquilinum, Rhinogobius similis, Silurus asotus, Solen gouldi, Struthiopteris nipponica, Thiara libertina, Turbo cornutus, Umbonium costatum, Viviparus malleatus
Wittliff, J.L.; Airth, R.L.
The extracellular thiaminase I of Bacillus thiaminolyticus. I. Purification and physicochemical properties
Biochemistry
7
736-744
1968
Paenibacillus thiaminolyticus, Paenibacillus thiaminolyticus M
Suzuki, K.; Ooba, J.I.
Reversible inactivation of cellular thiaminase I in Bacillus thiaminolyticus by thiamine and heteropyrithiamine
J. Biochem.
72
1053-1055
1972
Paenibacillus thiaminolyticus
Suzuki, K.; Ooba, J.I.
Reversible inactivation of extracellular thiaminase I in Bacillus thiaminolyticus. I. Inactivation by the primary substrate and reactivation by the secondary substrate
Biochim. Biophys. Acta
293
111-117
1973
Paenibacillus thiaminolyticus, Paenibacillus thiaminolyticus YUSM I00I
Agee, C.C.; Airth, R.L.
Reversible inactivation of thiaminase I of Bacillus thiaminolyticus by its primary substrate, thiamine
J. Bacteriol.
115
957-965
1973
Paenibacillus thiaminolyticus
Hutter, J.A.; Slama, J.T.
Inhibition of thiaminase I from Bacillus thiaminolyticus. Evidence supporting a covalent 1,6-dihydropyrimidinyl-enzyme intermediate
Biochemistry
26
1969-1973
1987
Paenibacillus thiaminolyticus, Paenibacillus thiaminolyticus BMM
Costello, C.A.; Kelleher, N.L.; Abe, M.; McLafferty, F.W.; Begley, T.P.
Mechanistic studies on thiaminase I. Overexpression and identification of the active site nucleophile
J. Biol. Chem.
271
3445-3452
1996
Paenibacillus thiaminolyticus (P45741), Paenibacillus thiaminolyticus
Kelleher, N.L.; Nicewonger, R.B.; Begley, A.P.; McLafferty, F.W.
Identification of modification sites in large biomolecules by stable isotope labeling and tandem high resolution mass spectrometry. The active site nucleophile of thiaminase I
J. Biol. Chem.
272
32215-32220
1997
Paenibacillus thiaminolyticus (P45741)
Ruml, T.; Silhankova, L.; Brunerova, M.
Purification of thiaminase I for analytical purposes
Potravinarske Vedy
13
181-187
1995
Paenibacillus thiaminolyticus, Paenibacillus thiaminolyticus DBM 1068
-
Campobasso, N.; Begun, J.; Costello, C.A.; Begley, T.P.; Ealick, S.E.
Crystallization and preliminary x-ray analysis of thiaminase I from Bacillus thiaminolyticus: space group change upon freezing of crystals
Acta Crystallogr. Sect. D
54
448-450
1998
Paenibacillus thiaminolyticus (P45741), Paenibacillus thiaminolyticus
Wu, M.; Papish, E.T.; Begley, T.P.
Mechanistic studies on thiaminase I. Identification of the product of thiamin degradation in the absence of the nucleophilic cosubstrate
Bioorg. Chem.
28
45-48
2000
Paenibacillus thiaminolyticus (P45741)
-
Hanes, J.W.; Kraft, C.E.; Begley, T.P.
An assay for thiaminase I in complex biological samples
Anal. Biochem.
368
33-38
2007
Paenibacillus thiaminolyticus, Paenibacillus thiaminolyticus (P45741)
Soriano, E.V.; Rajashankar, K.R.; Hanes, J.W.; Bale, S.; Begley, T.P.; Ealick, S.E.
Structural similarities between thiamin-binding protein and thiaminase-I suggest a common ancestor
Biochemistry
47
1346-1357
2008
Paenibacillus thiaminolyticus (P45741)
Liu, S.; Monks, N.R.; Hanes, J.W.; Begley, T.P.; Yu, H.; Moscow, J.A.
Sensitivity of breast cancer cell lines to recombinant thiaminase I
Cancer Chemother. Pharmacol.
66
171-179
2010
Paenibacillus thiaminolyticus
Liu, S.; Bae, Y.; Leggas, M.; Daily, A.; Bhatnagar, S.; Miriyala, S.; St Clair, D.K.; Moscow, J.A.
Pharmacologic properties of polyethylene glycol-modified Bacillus thiaminolyticus thiaminase I enzyme
J. Pharmacol. Exp. Ther.
341
775-783
2012
Paenibacillus thiaminolyticus
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