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PubMed2000

Some molecular and enzymatic properties of a homogeneous preparation of thiaminase I purified from carp liver.

A homogeneous preparation of thiaminase I (thiamine:base 2-methyl-4-aminopyrimidine-5-methenyl transferase, EC 2.5.1.2) was obtained from carp liver, for the first time from a nonbacterial source. Its molecular mass was 55 kDa by gel filtration and by SDS-PAGE regardless the presence of the reducing agent, indicating that the native enzyme consists of a single polypeptide chain. The determined sequence of 20 residues at the N-terminal of carp thiaminase I seemed to be unique. The enzyme was tested for ability to decompose a number of thiamine analogues. Even very extensive modifications of the thiazolium fragment were well tolerated, but around the pyrimidine fragment the active center seemed to exert steric restrictions against 1' (N)- and 2' (C)- atoms, while the 4'-amino group and untouched 6'-carbon atom were absolutely essential for the enzyme action. Numerous nucleophiles could be used by the enzyme as cosubstrates, aniline, pyridine, and 2-mercaptoethanol being the best among compounds tested. Protein chemical modification experiments indicated that histidine residues, carboxyl groups, and sulfhydryl groups may play specific roles in the thiaminase I-catalyzed reaction. Like in the bacterial enzyme, a sulfhydryl group may be a catalytically critical active-site nucleophile. The histidine residues and carboxyl groups may be essential for thiamine binding to the active site.

Alkyl and Aryl Transferases↗
PubMed1996

Mechanistic studies on thiaminase I. Overexpression and identification of the active site nucleophile.

Thiaminase I (EC 2.5.1.2) catalyzes the replacement of the thiazole moiety of thiamin with a wide variety of nucleophiles. Here we report the sequencing of a thiaminase I clone from Bacillus thiaminolyticus, the overexpression of the cloned gene in Escherichia coli, and the purification and characterization of the enzyme. Recombinant thiaminase I functions as a monomer with a Km for thiamin of 3.7 +/- 0.6 microM and a kcat of 34 s-1. Electrospray ionization Fourier-transform mass spectrometry identified a single sequencing error and demonstrated heterogeneity, finding molecular weights of 42,127, 42,198, and 42,255 due to added Ala and Gly-Ala at the amino terminus. Similar analysis of the 4-amino-2-methyl-6-chloropyrimidine inactivated enzyme indicated that the active site nucleophile involved in catalysis of the substitution reaction is located between Pro79 and Thr177. Subsequent cysteine-specific labeling and site-directed mutagenesis identified Cys113 as the active site nucleophile.

Alkyl and Aryl Transferases↗
PubMed1979

Cerebrocortical necrosis in ruminants: effect of thiaminase type 1-producing Clostridium sporogenes in lambs.

Large numbers of orally inoculated thiaminase type 1-producing Clostridium sporogenes failed to establish in the alimentary tract of two conventionally born lambs. Conversely, when similar inoculations were given to two gnotobiotic lambs, large populations of Cl sporogenes established in their rumens and correspondingly high levels of thiaminase were produced. No clinical symptoms of thiamine deficiency or cerebrocortical necrosis were seen despite the presence of high levels of thiaminase in the rumen of one of the gnotobiotic lambs for a period of 86 days.

Alkyl and Aryl Transferases↗
PubMed1975

Quantitative analysis of thiaminase activity in certain fish species.

Thiaminase I and II activity of Baltic herring, vendace, smelt and dace was measured. All four fish species were found to contain thiaminase activity. The amounts of thiaminase activity in mug of thiamine split per 100 g of fish tissue per hour at 37 degrees C were: in Baltic herring 115 +/- 60, in dace 11500 +/- 2050, in smelt 25 +/- 25 and in vendace 30+/- 15.

Animals↗
PubMedDate not supplied

[The presence of antithiamine factor--thiaminase--in organs of random-bred albino rats and mice].

Thiaminase activity was not found in rat and mice tissues (liver, kidney tissues) using radiometric procedure. Intact exogenous thiaminase I from bivalve freshwater mollusks was effectively detected in homogenates of rat or mice liver and kidney tissues within 6 hrs after the enzyme parenteral administration into these animals; at the same time, considerable amount of 14C-thiazol developed after thiaminase catalysis was estimated in urine.

Alkyl and Aryl Transferases↗
PubMedDate not supplied

[Antivitamin activity of thiaminase from B. thiaminolyticus with different routes of administration into animals].

Effect of bacterial thiaminase was studied in vivo after subcutaneous and intragastric administration of either the enzyme or of suspensions of intact and inactivated with an antibiotic bacterial cells, producing thiaminase. Activity of the thiamin-dependent enzymes (transketolase and alpha-ketoglutarate dehydrogenase) was distinctly decreased in liver and kidney tissues within 2 and 7 days after a single subcutaneous administration of 0.015 IU of the enzyme. Repeated administration of the enzyme within 4 days inhibited activity of thiamin-dependent enzymes in other tissues (heart, spleen, muscle and blood). Activity of thiamin-dependent enzymes in liver, kidney, heart, spleen, brain, muscles and blood was decreased more distinctly after administration of intact bacterial cells into mice stomach as compared to the effect of inactivated cells. Old animals were more sensitive to administration of intact bacterial cells than the young ones. The data obtained suggest that, besides the known thiamin-degrading effect of bacterial thiaminase in the intestinal contents, the enzyme exhibits functional activity within the animal tissues after the parenteral administration.

Alkyl and Aryl Transferases↗
PubMedDate not supplied

[The role of a cosubstrate in the kinetic mechanism of action of mollusk thiaminase I].

Mechanisms of interaction between thiaminase I and cosubstrate were studied using thiazole-2-14C-thiamine and 14C-nicotinic acid. The dissociation constant of nicotinic acid in the presence of the enzyme was of an order of 1 x 10(-6) M. A mechanism of the prestationary phase of a thiaminase reaction at -10 degrees was studied. The low temperature step, which demonstrated specific characteristics, was involved in analytic estimation of total thiamine concentration in vitro. Native thiaminase I from the bivalve mollusca Anodonta cygnea proved to be a holoenzyme.

Alkyl and Aryl Transferases↗
PubMed2005

Structural characterization of the regulatory proteins TenA and TenI from Bacillus subtilis and identification of TenA as a thiaminase II.

Bacillus subtilis gene products TenA and TenI have been implicated in regulating the production of extracellular proteases, but their role in the regulation process remains unclear. The structural characterization of these proteins was undertaken to help provide insight into their function. We have determined the structure of TenA alone and in complex with 4-amino-2-methyl-5-hydroxymethylpyrimidine, and we demonstrate that TenA is a thiaminase II. The TenA structure suggests that the degradation of thiamin by TenA likely proceeds via the same addition-elimination mechanism described for thiaminase I. Three active-site residues, Asp44, Cys135, and Glu205, are likely involved in substrate binding and catalysis based on the enzyme/product complex structure and the conservation of these residues within TenA sequences. We have also determined the structure of TenI. Although TenI shows significant structural homology to thiamin phosphate synthase, it has no known enzymatic function. The structure suggests that TenI is unable to bind thiamin phosphate, largely resulting from the presence of leucine at position 119, while the corresponding residue in thiamin phosphate synthase is glycine.

Alkyl and Aryl Transferases↗
PubMed1998

Crystal structure of thiaminase-I from Bacillus thiaminolyticus at 2.0 A resolution.

Thiaminase-I catalyzes the replacement of the thiazole moiety of thiamin with a wide variety of nucleophiles, such as pyridine, aniline, catechols, quinoline, and cysteine. The crystal structure of the enzyme from Bacillus thiaminolyticus was determined at 2.5 A resolution by multiple isomorphous replacement and refined to an R factor of 0.195 (Rfree = 0.272). Two other structures, one native and one containing a covalently bound inhibitor, were determined at 2.0 A resolution by molecular replacement from a second crystal form and were refined to R factors of 0.205 and 0.217 (Rfree = 0.255 and 0.263), respectively. The overall structure contains two alpha/beta-type domains separated by a large cleft. At the base of the cleft lies Cys113, previously identified as a key active site nucleophile. The structure with a covalently bound thiamin analogue, which functions as a mechanism-based inactivating agent, confirms the location of the active site. Glu241 appears to function as an active site base to increase the nucleophilicity of Cys113. The mutant Glu241Gln was made and shows no activity. Thiaminase-I shows no sequence identity to other proteins in the sequence databases, but the three-dimensional structure shows very high structural homology to the periplasmic binding proteins and the transferrins.

Alkyl and Aryl Transferases↗
PubMed1997

Identification of modification sites in large biomolecules by stable isotope labeling and tandem high resolution mass spectrometry. The active site nucleophile of thiaminase I.

A widely used procedure for site localization of covalent protein modifications involves proteolysis, partial chromatographic separation of the resulting complex mixture, and tandem mass spectrometry (MS/MS) to identify peptides whose molecular weight (Mr) has been increased appropriately by the modification. As found previously for MS of small molecules, this study shows that protein fragment identification can be greatly simplified by labeling the modification with stable isotopes. Further, the high resolution capabilities of Fourier transform MS make possible the direct identification of CH3/CD3-labeled peptides without chromatographic separation. Although separate Asp-N, Lys-C, and alpha-chymotrypsin digests of thiaminase I (42 kDa) yielded as many as 70 peptides, FTMS identification of the labeled peptide localized the modification site of a mechanism-based inhibitor to Arg101-Lys121, Asp90-Gly122, and Gly107-Tyr119, respectively. The measured mass difference values of the two labels agreed with that expected for CH3/CD3, 3.019 Da, with a standard deviation of 0.005 Da, providing persuasive identity verification. MS/MS fragmentation narrowed the site to Pro109-Phe118 and also caused loss of the derivative with a sulfur atom, uniquely identifying Cys113 as the thiaminase I active-site nucleophile among the 379 amino acids.

Alkyl and Aryl Transferases↗
PubMedDate not supplied

[Characteristics of the products of thiamine degradation by molluskan thiaminase I].

Tiaminase I (EC 2.5.1.2) catalyzed degradation of thiamin in the reaction mixture free of substrate-acceptor. Thiamin exhibited properties of both substrate-donor and -acceptor of the group transferred by thiaminase I. Identification of the products developed after thiaminase reaction involving thiamin was performed using chromatography and radiometry. Kinetics of the reaction is discussed.

Alkyl and Aryl Transferases↗
PubMed1987

Occurrence of thiaminase II in Saccharomyces cerevisiae.

It was found that cell-free extracts of Saccharomyces cerevisiae contain thiaminase II which hydrolyzes thiamine and thiamine analogs. The possible involvement of this enzyme and thiamine-synthesizing enzymes in thiamine production from thiamine antagonists is discussed.

Cytosol↗
PubMed1998

Crystallization and preliminary X-ray analysis of thiaminase I from Bacillus thiaminolyticus: space group change upon freezing of crystals.

Thiaminase I (Mr = 42 100) from B. thiaminolyticus, expressed in E. coli, has been crystallized by the vapor-diffusion method. Three crystal forms, two of which grew from 0.1 M sodium acetate (pH = 4.6), 0.2 M ammonium sulfate and 30%(w/v) PEG 2000, have been examined by X-ray analysis. One crystal form diffracted to 2.5 A at room temperature, was orthorhombic, and had unit-cell edges of a = 87.7, b = 120.5 and c = 76.7 A with space group P212121. A self-Patterson map showed a strong peak indicating noncrystallographic translational pseudosymmetry with (u, v, w) = (0.03, 0.0, 0.5). When these crystals were frozen at liquid-nitrogen temperatures, a second crystal form was observed which had unit-cell dimensions a = 85.5, b = 117.5 and c = 36.6 A with space group P21212. A third crystal form grew from 0.1 M Tris (pH = 8.5), 0.2 M sodium acetate trihydrate and 28%(w/v) PEG 6000 to produce orthorhombic crystals of space group P212121 with cell edges of a = 114.4, b = 123.1 and c = 92.5 A.

Alkyl and Aryl Transferases↗
PubMedDate not supplied

[Approaches to assessing dynamics of permeability and duration of thiaminase I action, administered parenterally to animals].

Two approaches and direct isotopic assay were employed to examine the time course of permeability and duration of action of tissue thiaminase I injected parenterally to albino mice. The injected enzyme was found to be detectable in renal and hepatic tissues 3 hours later. The action of the enzyme dermally injected in a dose of 1.2 IU/kg in the same tissues lasted 4 days. Experimental findings led to the conclusion that the thiamine degradation enzyme may be used in experimental biology in order to estimate the duration of its action and the time course of gross molecular permeability in the tissue of test animals.

Alkyl and Aryl Transferases↗
PubMed1954

Thiaminase.

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Enzymes↗