Enzymatic conversion of the antibiotic metronidazole to an analog of thiamine

We propose that adverse effects of the antibiotic metronidazole may be due, wholly or in part, to its conversion to a thiamine analog and consequent vitamin B1 antagonism. Consistent with this hypothesis, the drug is accepted as a substrate for the thiaminase (EC 2.5.1.2) elaborated as an exoenzyme by the human gut flora constituent Bacillus thiaminolyticus and is also a substrate for the intracellular thiaminase of the mollusk Venus mercenaria. The product, identified as the 1-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-3-(2-hydroxyethyl)-2-methyl-4 - nitroimidazolium cation, is a close structural analog of thiamine and is an effective inhibitor of thiamine pyrophosphokinase in vitro. Due to its susceptibility to nucleophilic attack, the analog is unstable, releasing inorganic nitrite under mild conditions. Enzymatic alkylation reactions such as that effected by thiaminase may have general pharmacological significance as a route of increasing the electrophilicity and/or reduction potential of drugs which are heterocyclic weak bases.     

Possible functional roles of carboxyl and histidine residues in a soluble thiamine-binding protein of Saccharomyces cerevisiae

The reaction of a soluble thiamine-binding protein of Saccharomyces cerevisiae with water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, at pH 4.5, results in a remarkable loss of its binding activity with thiamine. Thiamine above 0.1 mM substantially protects the protein against this inactivation. In addition to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, the thiamine-binding protein is also inactivated by diethylpyrocarbonate. The inactivation is time-dependent and follows second-order kinetics. Restoration of the binding activity by incubation of inactivated protein with hydroxylamine was observed. thiamine and pyrithiamine are effective to prevent the inactivation. From these results it is strongly suggested that both the carboxyl and the histidine residues in the protein are involved in the binding site for thiamine. It is proposed that the binding involves interactions between charged groups on the protein with the quaternary nitrogen of the thiazolium moiety and with the basic ring nitrogen of the pyrimidine moiety in thiamine molecule.     

Bracken thiaminase-mediated neurotoxic syndromes

The toxicity of bracken ( Pteridium aquilinum (L.) Kuhn) to animals is complicated because this plant elaborates more than one type of agent harmful to livestock. An enzyme, thiaminase I, which destroys thiamine, is responsible for the neurotoxic syndrome. Using a radiochemical assay, the distribution of thiaminase I activity in bracken throughout the growing season has been ascertained: levels are high in the rhizome and young buds, but fall sharply in the fronds as the aerial parts of the plant unfold.
The so-called thermostable 'antithiamine' factors present in bracken and other plant species are discussed.
The biochemical lesions of thiamine deficiency in animals are briefly outlined, and the clinical syndrome caused by the inclusion of bracken fronds or rhizomes in the diet for simple-stomached animals (rat, horse, pig) and a ruminant (sheep) are described.
All these neurotoxic syndromes respond to thiamine therapy in a dramatic way, if administered during the early stages of the disease.     

Metabolic Effects of Acute Thiamine Depletion Are Reversed by Rapamycin in Breast and Leukemia Cells

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. Immunoblot analyses showed corresponding differences in expression of BCKDH pathway enzymes, and partial protection of thiaminase growth inhibition by gabapentin indicated that BCKDH inhibition may be a mechanism of thiaminase-mediated toxicity. Surprisingly, most of thiaminase-mediated metabolomic effects were also reversed by rapamycin. Thus, these studies demonstrate that acute intracellular thiamine depletion by recombinant thiaminase results in metabolic changes in thiamine-dependent metabolism, and demonstrate a previously unrecognized role of mTOR signaling in the regulation of thiamine-dependent metabolism.     

Cell-Bound Thiaminase I of Bacillus thiaminolyticus

The distribution of the extracellular enzyme, thiaminase I, was determined for logarithmically growing cultures of Bacillus thiaminolyticus. About 60% of the enzyme is associated with the cells throughout the growth cycle. The remainder of the enzyme is in the culture medium. The release of the cell-bound thiaminase I is examined under a variety of conditions. The rate and extent of release is dependent on the pH and the nature of the incubation solution. The release process appears to be relatively independent of de novo protein synthesis, energy derived from oxidative phosphorylation, or divalent metal ions. The absence of carbon or nitrogen sources has little effect on the release of the enzyme. Cell-bound thiaminase I probably is the immediate precursor for extracellular thiaminase I found in the culture medium. Washed cells continue to release thiaminase I at the expense of cell-bound enzyme. In addition, purified cell-bound thiaminase I is indistinguishable from purified extracellular thiaminase I by a number of physical and kinetic criteria.     

Investigation of essential SH-groups of bacterial formate dehydrogenase]

Modification of two SH-groups in the molecule of formate dehydrogenase by dithiobisnitrobenzoate or to dacetamide results in the enzyme inactivation. Coenzymes, but not the substrate, protect the enzyme against the inactivation. NAD in the presence of potassium azide completely preserves the enzyme activity. Two SH-groups per enzyme molecule are protected from modification. The Km values for partially inactivated formate dehydrogenase remain constant for both substrates. The enzyme with modified SH-groups does not bind conezymes. The pH-dependence of the inactivation rate reveals the ionizable group with pK 9.6 (25 degrees C). The involvement of essential SH-groups in coenzyme binding is discussed.     

Thiaminase activity of gastrointestinal contents of salmon and herring from the Baltic Sea

Potential thiaminase activity of Baltic herring Clupea harengus ranged from 0 to c. 55 nmol g-1 min-1 while potential thiaminase activity in Baltic salmon Salmo salar gastrointestinal (GI) contents ranged from 7 to c. 60 nmol g-1 min-1. About 30% of the Baltic herring analysed had a potential thiaminase activity equivalent to Baltic salmon GI contents. The results are consistent with the hypothesis that thiaminase in the forage fish of Baltic salmon may be an important link in the aetiology of the thiamine deficiency syndrome, M74, in Baltic salmon and indicate that Baltic salmon might feed selectively on Baltic herring with high thiaminase activity.     

Multiple modes of catalysis-dependent inhibition and inactivation of aortic lysyl oxidase

Lysyl oxidase purified from bovine aorta can oxidize simple alkyl mono- and diamine substrates yielding the respective aldehyde, H2O2, and ammonia as products. The oxidation of such substrates is limited to approximately 100 catalytic turnovers per enzyme molecule since lysyl oxidase is syncatalytically and irreversibly inactivated in the course of oxidation of these amines. The present study reveals that addition of oxidant scavengers protects significantly against inactivation of lysyl oxidase and that the ammonia product is a reversible competitive inhibitor of amine oxidation. Further, the enzyme becomes covalently labeled by the amine substrate or its enzyme-processed derivative during catalysis. Thus, lysyl oxidase appears subject to multiple modes of catalysis-dependent inhibition or inactivation. Syncatalytic inactivation of lysyl oxidase might represent a means of restricting the activity of this enzyme toward its elastin and collagen substrates in vivo.     

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.     

Regulation of pea mitochondrial pyruvate dehydrogenase complex activity: inhibition of ATP-dependent inactivation

In contrast to the pyruvate dehydrogenase complex (PDC) from animal mitochondria, our in situ and in vitro studies indicate that the ATP:ADP ratio has little or no effect in regulating the mitochondrial pyruvate dehydrogenase complex from green pea seedlings. Pyruvate was a competitive inhibitor of ATP-dependent inactivation (Ki = 59 microM), while the PDC had a Km for pyruvate of microM. Thiamine pyrophosphate, the coenzyme for the pyruvate dehydrogenase (PDH) component of the complex, did not inhibit ATP-dependent inactivation when used alone but it enhanced inhibition by pyruvate. As such, thiamine pyrophosphate was a competitive inhibitor (Ki = 130 nM) of ATP-dependent inactivation. A model is proposed for the pyruvate plus thiamine pyrophosphate inhibition of ATP-dependent inactivation of the pyruvate dehydrogenase complex in which pyruvate exerts its inhibition of inactivation by altering or protecting the protein substrate from phosphorylation and not by directly inhibiting PDH kinase.     

Functional carboxylic group in the active center of transketolase

Baker's yeast transketolase is rapidly inactivated in the presence of carboxylic group modifiers, i.e., 1-ethyl-3(3'-dimethylaminopropyl)-carbodiimide or Woodward's reagent K. This inactivation is due to modification of the carboxylic group in the enzyme active center. The essential groups localized in the two active centers of transketolase differ in the rate of modification; accordingly, the inactivation kinetics appears as biphasic. A complete loss of the enzyme activity occurs as a result of modification of one carboxylic group per enzyme active center. The pKa value of modifiable groups is equal to about 6.5. This modification decreases by two orders of magnitude the affinity of the substrate for the active center. The carboxylic groups are not directly involved in the interaction with the substrates; their modification does not significantly affect the coenzyme binding. It is supposed that these groups are responsible for the deprotonation of the second carbon in the thiamine pyrophosphate thiazolium ring.     

Chronic alcoholism in rats induces a compensatory response, preserving brain thiamine diphosphate, but the brain 2-oxo acid dehydrogenases are inactivated despite unchanged coenzyme levels

Thiamine-dependent changes in alcoholic brain were studied using a rat model. Brain thiamine and its mono- and diphosphates were not reduced after 20 weeks of alcohol exposure. However, alcoholism increased both synaptosomal thiamine uptake and thiamine diphosphate synthesis in brain, pointing to mechanisms preserving thiamine diphosphate in the alcoholic brain. In spite of the unchanged level of the coenzyme thiamine diphosphate, activities of the mitochondrial 2-oxoglutarate and pyruvate dehydrogenase complexes decreased in alcoholic brain. The inactivation of pyruvate dehydrogenase complex was caused by its increased phosphorylation. The inactivation of 2-oxoglutarate dehydrogenase complex (OGDHC) correlated with a decrease in free thiols resulting from an elevation of reactive oxygen species. Abstinence from alcohol following exposure to alcohol reactivated OGDHC along with restoration of the free thiol content. However, restoration of enzyme activity occurred before normalization of reactive oxygen species levels. Hence, the redox status of cellular thiols mediates the action of oxidative stress on OGDHC in alcoholic brain. As a result, upon chronic alcohol consumption, physiological mechanisms to counteract the thiamine deficiency and silence pyruvate dehydrogenase are activated in rat brain, whereas OGDHC is inactivated due to impaired antioxidant ability.     

Thiaminase activity of Baltic salmon prey species: a comparision of net- and predator-caught samples

Thiaminase activity was determined for Gulf of Bothnia (GB) and Gulf of Finland (GF) Baltic herring Clupea harengus membras , sprat Sprattus sprattus and three-spined stickleback Gasterosteus aculeatus sampled from either trawl or gillnet catches or from Baltic salmon Salmo salar stomachs. The thiaminase activity in Baltic herring was about 10-fold higher than that in sprat, and there was almost no thiaminase activity in three-spined stickleback. Thiaminase activity of undigested Baltic herring found in Baltic salmon stomachs was significantly higher than that of trawl-caught Baltic herring from the same sea area, suggesting that there may be a higher risk of predation for Baltic herring with high thiaminase activity, possibly linked to their health. Thiaminase activity of the gastrointestinal contents of Baltic salmon, feeding almost entirely on Baltic herring in the GB, was significantly higher than for Baltic salmon feeding on both Baltic herring and sprat in the GF. Therefore, Baltic herring may be the major source of thiaminase for Baltic salmon. A tank experiment demonstrated that thiaminase activity in Baltic herring may vary, even within very short time periods. The results were consistent with the hypothesis that the thiaminase content in Baltic salmon forage fish may be an important link in the aetiology of the thiamine deficiency syndrome, M74, in Baltic salmon.     

Inhibition of thiaminase I from Bacillus thiaminolyticus. Evidence supporting a covalent 1,6-dihydropyrimidinyl-enzyme intermediate

Thiaminase I from Bacillus thiaminolyticus strain Matsukawa et Misawa is completely and irreversibly inhibited by treatment with 4-amino-6-chloro-2-methylpyrimidine. Inhibition is a time-dependent first-order process, exhibiting a half-time of 4 h at an inhibitor concentration of 5 mM. A specific active-site-directed inactivation is supported by protection of the enzymatic activity in the presence of the substrates thiamin and quinoline as well as by the observation that a stoichiometric amount of inorganic chloride is released during inactivation. 4-Amino-5-(anilinomethyl)-6-chloro-2-methylpyrimidine, which resembles the structure of the product of base exchange of thiamin with aniline, inactivates thiaminase approximately 2 orders of magnitude faster. Inactivation is again complete and irreversible and is a time-dependent first-order process, in this case exhibiting saturation at low inhibitor concentrations (KI = 96 microM). Enzyme inactivation can be explained as the result of displacement of chloride from the chloropyrimidine by a nucleophile at the enzyme active site. The inactivation suggests that the Zoltewicz-Kauffman model of bisulfite-catalyzed thiamin cleavage [Zoltewicz, J. A., & Kauffman, G. M. (1977) J. Am. Chem. Soc. 99, 3134-3142], which calls for the reversible nucleophilic addition of catalyst across the 1,6 double bond of thiamin's pyrimidine ring, may be applicable to thiaminase as well.     

Modification of arginine residues in pyruvate kinase (author's transl)

Pyruvate kinase from pig heart is inactivated by the specific arginyl reagent phenylglyoxal. The loss of activity is caused by the reaction of a single molecule of phenylglyoxal per subunit of enzyme. During inactivation 3 - 6 arginyl residues are modified dependent on the concentration of phenylglyoxal used for modification. The solubility of the protein is reduced by the modification. ATP or phosphoenolpyruvate protect against inactivation. A single arginine is less subject to chemical modification in their presence. Therefore we assume that an arginine is essential at the substrate binding site. The activating ion K does not affectinactivation, where as Mg2 diminishes inactivation. Pyruvate kinase from rabbit muscle is modified by phenylglyoxal in a similar manner.     

The importance of Δ1-pyrroline in the aetiology of cerebrocortical necrosis

Bacterial thiaminase I associated with cerebrocortical necrosis of cattle and sheep is shown to utilise Δ¹-pyrroline and related compounds as cosubstrates. The product resulting from the reaction of thiamine and Δ¹-pyrroline is 1-(4-amino-2-methylpyrimidin-5-ylmethyl)-1-pyrrolinium chloride. This compound has been identified in the brains of calves suffering from cerebrocortical necrosis. The implications of these findings in the aetiology of other thiamine-responsive diseases of the central nervous system are briefly discussed.     

Diphtheria toxin: the effect of nitration and reductive methylation on enzymatic activity and toxicity.

Diphtheria toxin is rapidly inactivated upon reaction with tetranitromethane. inactivation is partially prevented in the presence of the substrate NAD. The loss of enzymatic activity and of toxicity is concomitant with the modification of one tyrosyl residue per molecule, located in the fragment A. Completely inactivated toxin (more than 5 nitrotyrosines per molecule) is a good toxin antagonist for HeLa cells binding sites indicating that the integrity of its fragment B is preserved. Methylation of lysyl residues leads to a decrease of toxicity and enzymatic activity but only after the modification of about 20 lysines per molecule. This methylated toxin however can still bind NAD and seems to possess a functional fragment B. Enzymatic site of diphtheria toxin fragment A seems thus to contain one essential tyrosyl residue implicated in the binding of NAD and at least one lysine not implicated in this dinucleotide binding.     

Cobalt(III) labeling of methionyl-tRNA synthetase from Escherichia coli.

Native and trypsin-modified methionyl-tRNA synthetases from Escherichia coli were found to be inactivated by incubation in the presence of Co(III) complexes of ATP, stabilized either by imidazole or phenanthroline, or by oxidation in situ to Co(III) of the substrate ATP-Co(II). It has been shown that the inactivation proceeds by specific labeling of the catalytic ATP-Mg(II) site of the synthetases. The enzymes are completely inactivated by the incorporation of one cobalt atom and one ATP molecule per active site. The inactivated enzymes may be stored for a long period without significant reactivation or removal of the cobalt label. In the presence of dithiothreitol or 2-mercaptoethanol, the labeled enzymes recover full activity with concomittant release of the bound label molecules.     

Evidence for an aldehyde intermediate in the catalytic mechanism of thiamine oxidase

Thiamine oxidase catalyzes the four-electron oxidation of the 5-hydroxyethyl group of thiamine to form thiamine acetic acid via an aldehyde intermediate. Evidence for the formation of this intermediate is derived from a number of kinetic approaches. The rate of thiamine acetic acid formation, as monitored by the rate of proton release, is subject to substrate inhibition and to inhibition by the presence of semicarbazide while the rate of O2 consumption (due to thiamine oxidation to the aldehyde and subsequently to the carboxylic acid) is unaffected. The transient formation of an intermediate with a maximal absorption at 370 nm in stopped-flow turnover experiments is dependent on the pH and the substrate concentration, and is prevented by the presence of semicarbazide, thus suggesting this transient absorption intermediate to be a result of formation of the aldehyde intermediate. A similar spectral intermediate is observed when hydroxythiamine is the substrate but is not observed with pyrithiamine. In the presence of large concentrations of pyrithiamine, the enzyme undergoes an irreversible inactivation which is not reversed on removal of pyrithiamine or its oxidation products by gel filtration or dialysis. This inhibition is prevented by the presence of thiols or of semicarbazide and is suggested to be due to the release of the aldehyde form of pyrithiamine from the catalytic site, which then reacts with the enzyme in a nonspecific manner. The structure of the 370-nm-absorbing intermediate is currently unknown but is suggested not to be the "yellow form" of thiamine. This suggestion is due to observed differences in absorption spectral properties and to the fact that it can also be formed from hydroxythiamine, which does not form the "yellow form" of thiamine on alkaline treatment. Taken together, these data suggest that, at or below saturating concentrations, thiamine remains bound to the catalytic site during the two sequential two-electron transfer steps, with 2 mol O2 being reduced to 2 mol H2O2. At high concentrations (greater than 10 Km), the intermediate thiamine aldehyde can be displaced from the catalytic site by thiamine simply by a mass-action effect.     

Modification of glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides with the 2',3'-dialdehyde derivative of NADP+ (oNADP+)

Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides is irreversibly inactivated by the 2,3'-dialdehyde of NADP+ (oNADP+) in the absence of substrate. The inactivation is first order with respect to NADP+ concentration and follows saturation kinetics, indicating that the enzyme initially forms a reversible complex with the inhibitor followed by covalent modification (KI = 1.8 mM). NADP+ and NAD+ protect the enzyme from inactivation by oNADP+. The pK of inactivation is 8.1. oNADP+ is an effective coenzyme in assays of glucose-6-phosphate dehydrogenase (Km = 200 microM). Kinetic evidence and binding studies with [14C] oNADP+ indicate that one molecule of oNADP+ binds per subunit of glucose-6-phosphate dehydrogenase when the enzyme is completely inactivated. The interaction between oNADP+ and the enzyme does not generate a Schiff's base, or a conjugated Schiff's base, but the data are consistent with the formation of a dihydroxymorpholino derivative.