Biosynthesis of Thiazole in Exobasidium

In some Exohmidium strains, which grow on a synthetic medium containing glucose citralc, and thiamine as the only organic compounds, growth usually does not start until after more than 800 honrs if thiamine is replaced by pyrimidine. Homocysteine and methionine are able to shorten this lag phase of E. vaccinil“myrt.” Methionine is growth-promoting only at about pH 4. At low concentrations the yield is proportional to the concentration of methionine. Growth ceases when the methionine is consumed and the fungus then enters a lag phase as long as that in methionine-free medium. Methionine does not indnce the thiazole-synthesizing enzymes, but is probably consumed as a precursor of thiazole. The utilization of this precursor is delayed by some growth factors and amino acids, i.e. cholin, betaine, glutamic acid or glutamine. The delayed adaptation of the methionine- and thiazole-synthesizing enzymes seems to be regulated by changes in the nutritional state of the ageing cultures. The mechanism of this regulation has not been settled.     

GENETIC BLOCKS IN THE THIAMINE SYNTHESIS OF THE ANGIOSPERM ARABIDOPSIS,,

Five thiamine-requiring mutants were obtained at two loci. Two are blocked in the synthesis of the pyrimidine part of the vitamin, the other three have lost the ability to make the thiazole moiety. None of the tested substances suggested as possible or likely precursors of the pyrimidine or the thiazole components of thiamine displayed any activity in the mutants. These conditional lethals responded to remarkably small supplements of thiamine. The pyrimidine-requiring mutants utilized to some extent the anti-vitamin neopyrithiamine. The thiazole-less mutants grew on basal media supplemented only with the analog, oxythiamine. Thiamine deficiency, irrespective of the position of the genetic block in the synthesis, results in a characteristic anomaly of pigmentation. The position of the py locus in the second linkage group has been determined. Allelic complementation has not been detected. The frequency of mutations affecting thiamine synthesis appears about the same in Arabidopsis as in fungi. The general frequency of reparable genetic lesions is, however, one to two orders of magnitude lower in Arabidopsis than that in fungi or bacteria.     

Effect of water soluble vitamins and their analogues on growth of Candida albicans II. Vitamin B12, thiamine,oxythiamine, neopyrithiamine,substituted pyrimidines and thiazoles

Summary By employing wide ranges in vitamin concentrations in biotin basal mineral synthetic medium, it was demonstrated that vitamin B₁₂ markedly stimulated the growth ofCandida albicans, the organism showing a partial dependency upon this vitamin. Growth inhibition by 5-fluorouracil was reversed non-competitively by vitamin B₁₂, suggesting that B₁₂ has a role in nucleic acid biosynthesis of the organism. Thiamine was growth stimulatory, the organism being partially dependent upon this vitamin as well. Neopyrithiamine and oxythiamine were growth inhibitory in thiamine-free biotin basal mineral synthetic medium although the halves of each inhibitor compound were non-inhibitory. Neopyrithiamine inhibition was reversed by intact thiamine but not by pyrimidine thiamine or thiazole thiamine; while oxythiamine inhibition was reversed by thiamine and pyrimidine thiamine but not by thiazole thiamine, the inference being drawn that oxythiamine selectively blocks utilization of pyrimidine thiamine. Twenty-seven different substituted pyrimidines, thiazoles and related thiamine compounds were all utilizable byC. albicans in thiamine-free basal synthetic mineral medium, the organism presumably synthesizing thiamine when presented with the constituent parts of these thiamine analogues. Substitution of sulfur of the thiazole ring with oxygen, as in -methyloxazolium, failed to produce an inhibitory compound forC. albicans. Acetylthiamine, allithiamine, cocarboxylase, tetrahydrothiamine and dihydrothiamine were equally as growth stimulatory as thiamine.     

Observations on the biosynthesis of thiamine in yeast

1. Methods are described for the isolation of radioactively pure thiamine from yeast and its degradation on a small scale to its cyclic components. 2. A degradation of the pyrimidine ring and a thin-layer method for the separation of thiamine, its derivatives and pyrimidine and thiazole residues are described. 3. [(14)C]Formate is more effectively incorporated into the pyrimidine residue than into the thiazole residue, whereas the reverse is true with l-[Me-(14)C]methionine. 4. Experiments with [Me-(14)C,(35)S]methionine demonstrate that methionine provides an intact unit for the biosynthesis of the thiazole ring. 5. [6-(14)C]Orotic acid is insignificantly incorporated into the pyrimidine residue of thiamine. 6. Experiments with [1-(14)C]- and [2-(14)C]-acetate indicate that it is incorporated as a unit into the thiazole residue, but that only C-2 is incorporated into the pyrimidine residue. 7. l-[U-(14)C]Alanine is also effectively incorporated into the thiazole residue. 8. These results are discussed in relation to possible pathways of biosynthesis of the two ring components of the thiamine molecule.     

The control mechanism of thiamine biosynthesis. A model for the study of control of converging pathways

1. Thiamine or the pyrimidine moiety of thiamine added in excess to a growing culture of Salmonella typhimurium LT2 repressed subsequent thiamine synthesis in non-growing organisms. 2. A mutant unable to convert added pyrimidine moiety into thiamine was not repressible by the pyrimidine, showing that thiamine, not the pyrimidine, was the repressor. 3. Thiamine repression occurred at 40mmug. of thiamine/mg. dry wt. or above and de-repression occurred at 30mmug. of thiamine/mg. dry wt. or below. 4. Thiamine controlled the pyrimidine and thiazole pathways at the same concentration and to the same extent. 5. Biosynthesis of the thiazole moiety had, in contrast with biosynthesis of the pyrimidine moiety, an additional feedback inhibition control that allowed utilization of the exogenous thiazole. 6. The enzymes joining the pyrimidine and thiazole moieties were repressible by high concentrations of thiamine. 7. Thiamine was rapidly converted into thiamine pyrophosphate and this appeared to be the active repressor. 8. Theoretical aspects of control of converging pathways are discussed.     

Pathway of thiamine pyrophosphate synthesis in Micrococcus denitrificans.

The pathway of thiamine pyrophosphate (TPP) biosynthesis, which is formed either from exogeneously added thiamine or from the pyrimidine and thiazole moieties of thiamine, in Micrococcus denitrificans was investigated. The following indirect evidence shows that thiamine pyrophosphokinase (EC 2.7.6.2) catalyzes the synthesis of TPP from thiamine: (i) [35S]thiamine incubated with cells of this microorganism was detected in the form of [35S]thiamine; (ii) thiamine gave a much faster rate of TPP synthesis than thiamine monophosphate (TMP) when determined with the extracts; and (iii) a partially purified preparation of the extracts can use thiamine, but not TMP, as the substrate. The activities of the four enzymes involved in TMP synthesis from pyrimidine and thiazole moieties of thiamine were detected in the extracts of M. denitrificans. The extracts contained a high activity of the phosphatase, probably specific for TMP. After M. denitrificans cells were grown on a minimal medium containing 3 mM adenosine, which causes derepression of de novo thiamine biosynthesis in Escherichia coli, the activities of the four enzymes involved with TMP synthesis, the TMP phosphatase, and the thiamine pyrophosphokinase were enhanced two- to threefold. These results indicate that TPP is synthesized directly from thiamine without forming TMP as an intermediate and that de novo synthesis of TPP from the pyrimidine and thiazole moieties involves the formation of TMP, followed by hydrolysis to thiamine, which is then converted to TPP directly. Thus, the pathway of TPP synthesis from TMP synthesized de novo in M. denitrificans is different from that found in E. coli, in which TMP synthesized de novo is converted directly to TPP without producing thiamine.     

The de-repression of thiamine biosynthesis by adenosine. A tool for investigating this biosynthetic pathway

1. Growth of Salmonella typhimurium LT2 in the presence of adenosine was shown to cause enormous synthesis of thiamine in washed-cell suspensions. 2. Evidence that this was due to de-repression and not an accumulation of precursors was obtained by using a mutant blocked in the biosynthesis of the thiazole moiety, which showed a similarly large synthesis of the pyrimidine of thiamine. 3. The specific requirements for a source of energy, nitrogen and sulphur were investigated, and indicated new synthesis in this system.     

Effect of Glycine on Thiazole Biosynthesis in Escherichia coli

Glycine was found to replace thiamine thiazole for the growth of the thiazoleless mutant of Escherichia coli; it also stimulated the production of thiamine thiazole by washed cell suspensions of the mutant.     

Gene locus specificity of the glucose effect in the thiamine pathway of the angiosperm, Arabidopsis

All mutants at 3 loci in Arabidopsis thaliana (L.) Heynh., a higher plant, that are associated with the synthesis or coupling of the thiazole moiety of thiamine are susceptible to reversible glucose inhibition. In contrast, several different alleles involved in the synthesis of the pyrimidine moiety of the vitamin are insensitive to glucose. Glucose and maltose are equally effective inhibitors while fructose, lactose, ribose, and xylose are toxic. This toxicity is not released by added thiamine.     

Neurospora crassa CyPBP37: a cytosolic stress protein that is able to replace yeast Thi4p function in the synthesis of vitamin B1

Recently, we identified CyPBP37 of Neurospora crassa as a binding partner of cyclophilin41. CyPBP37 function had not yet been described, although orthologs in other organisms have been implicated in the biosynthesis of the thiazole moiety of thiamine (vitamin B1) and/or stress-related pathways. Here, CyPBP37 is characterized as an abundant cytosolic protein with a functional NAD-binding site. Saccharomyces cerevisiae mutants lacking Thi4p (the CyPBP37 ortholog) are auxotrophic for vitamin B1 (thiamine) but can grow in the presence of the thiazole moiety of thiamine, suggesting a role for Thi4p in the biosynthesis of thiazole. N.crassa CyPBP37 is able to functionally replace Thi4p in yeast thiazole synthesis. Cellular fractionation studies revealed that Thi4p is a cytosolic protein in S.cerevisiae, like its ortholog CyPBP37 in N.crassa. This implies that thiamine synthesis takes place in the cytosol of both organisms and not in the mitochondria, as suggested. The expression of CyPBP37 and Thi4p is repressed by thiamine but not by thiazole in the growth medium. In addition to its function in thiazole synthesis, CyPBP37 is a stress-inducible protein. N.crassa cyclophilin41 can chaperone the folding of CyPBP37, its own binding partner.     

ORCHID MYCORRHIZA: VITAMIN PRODUCTION AND REQUIREMENTS BY THE SYMBIONTS

A Rhizoctonia species isolated from Cymbidium has been cultured successfully on a defined medium consisting of minerals, sugar, thiamine, and folic acid. Thiamine can be replaced by its thiazole component, which is probably produced by germinating orchids. The fungus apparently produces the pyrimidine moiety of thiamine, a compound which may enhance growth of certain orchid seedlings. Niacin is also provided by the fungus. Para-aminobenzoic acid, a constituent of folic acid, produced and released by orchid seeds, can satisfy the vitamin requirement of the fungus. These findings point to the possibility that orchids and their fungi may have coevolved with respect to vitamin requirements. The data also suggest that exchanges of vitamins or their components between orchids and endophytes are important aspects of the symbiotic relationship.     

Biosynthesis of thiamine. Incorporation experiments with 14C-labelled substrates and with [15N]glycine in Saccharomyces cerevisiae

1. Yeast was grown in a minimal synthetic medium together with a range of (14)C-labelled substrates under standardized conditions. After isolation, the purified thiamine was cleaved by sulphite and the pyrimidine and thiazole moieties were purified and assayed for radioactivity. 2. In order of decreasing incorporation, [(14)C]formate, [3-(14)C]serine, [2-(14)C]glycine and [2-(14)C]acetate supplied label for the pyrimidine, and [2-(14)C]glycine, [3-(14)C]serine, [1-(14)C]glycine, [(14)C]formate and [2-(14)C]acetate for the thiazole. Incorporation of label into the fragments from several other (14)C-labelled substrates, including [Me-(14)C]- and [3,4-(14)C(2)]-methionine, was insignificant. 3. [3-(14)C]Serine was shown not to contribute label to C-2 of the thiazole ring. 4. Significant incorporation of nitrogen from [(15)N]glycine into the thiazole moiety, but not into the pyrimidine moiety, was established. 5. It appears that C-2 and N-3 of the thiazole ring are formed from C-2 and the nitrogen atom of glycine, but the entire methionine molecule does not appear to be implicated.     

Precursors of the pyrimidine moiety of thiamine

1. A method was devised for obtaining the pyrimidine moiety of thiamine in a pure form after its excretion into the medium by de-repressed washed-cell suspensions of mutants of Salmonella typhimurium LT2. 2. By using amino acid-requiring mutants, this excretion of pyrimidine moiety was shown to be dependent on the presence of both methionine and glycine. 3. In the presence of either [Me-¹⁴C]methionine or [G-¹⁴C]methionine, methionine-requiring mutants did not incorporate radioactivity into the pyrimidine moiety. 4. In contrast, both [1-¹⁴C]glycine and [2-¹⁴C]glycine were incorporated into the pyrimidine moiety excreted by glycine-requiring mutants, and this occurred with little or no dilution of specific radioactivity. 5. The possible requirement for methionine as a cofactor and the significance of the incorporation of both carbon atoms of glycine are discussed.     

Biosynthesis of thiazole from thiamine in Escherichia coli]

The incorporation into the thiazole moiety of thiamine of several labeled compounds has been studied on short time incubations of washed-cells suspensions. No incorporation of radioactivity from [G-14C] methionine was found in a mutant auxotrophic for methionine. No radioactivity was incorporated from [U-14C] aspartate or from [U-14C] serine. The incorporation of 35S from sulphate was lowered by cysteine or glutathione but was unaffected by methionine or homocystine. Although the synthesis of thiazole is dependent on methionine, neither the sulphur atom nor the carbon chain of thiazole originate from methonine in E. coli. No carbon originates from cysteine which is the likely direct donor of sulphur.     

Isolation of nutritional mutants inArabidopsis thaliana

An accumulation method was devised for the isolation of nutritional mutants of the CruciferArabidopsis thaliana. Six mutants were obtained, in all of which the synthesis of thiamine was blocked. The mutants showed chlorophyll deficiencies and were more or less depressed in growth. Thiamine, when added to the substrate, fully restored normal development. Three of the mutants also grew on a substrate containing the pyrimidine moiety of the vitamin molecule, one on a medium with the thiazole part, one on a substrate containing a mixture of both compounds, whereas the sixth mutant required the complete thiamine molecule for normal growth. Two of the three pyrimidine-less mutants were allelic, the third, when crossed with each of the other two, yielded F₁'s showing wild type growth in their youth but deficiency symptoms in later stages. In each of the other mutants different loci were involved. The relation between the method employed and the type of mutant isolated is discussed.     

Cloning and regulation of Schizosaccharomyces pombe thi2, a gene involved in thiamine biosynthesis.

Biosyntheses of the pyrimidine and thiazole moieties of the thiamine molecule occur by separate pathways. In Schizosaccharomyces pombe, a gene, thi2, is responsible for thiazole synthesis [Schweingruber et al., Curr. Genet. 19 (1991) 249-254]. We have cloned a 3.1-kb genomic S. pombe fragment which can functionally complement a thi2 mutant. The fragment maps genetically at the thi2 site, indicating that it carries thi2. As shown by Northern hybridization analysis, the appearance of thi2 mRNA levels is repressed when cells are grown in the presence of thiamine and 5-(2-hydroxyethyl)-4-methylthiazole. The thi3 gene involved in the biosynthesis of the pyrimidine moiety, is also regulated by thiamine [Maundrell, J. Biol. Chem. 265 (1990) 10857-10864; Schweingruber et al., Curr. Genet. 19 (1991) 249-254]. We previously identified and analyzed four regulatory genes (tnr1, tnr2, tnr3, and thi1) that are responsible for the regulation of thi3 [Schweingruber et al., Genetics (1992) in press]. Mutants defective in these regulatory genes affect expression of thi2 in a similar way to thi3. This indicates that biosynthesis of the pyrimidine and thiazole moieties are under common genetic control in S. pombe.     

The nature of the requirement of Saccharomyces carlsbergensis for vitamin B6

Saccharomyces carlsbergensis 4228, an organism widely used for determination of vitamin B₆, grows well without this vitamin if thiamine is also omitted from the basal medium, and an inoculum grown in a thiamine-low medium is used. Thiamine inhibits growth when added to such a medium. The thiazole moiety of thiamine, but not the pyrimidine, is also inhibitory, but less so than thiamine itself.Growth inhibition by thiamine is prevented by vitamin B₆. At low concentrations of thiamine, the amount of vitamin B₆ required for growth increases with the thiamine concentration; at concentrations of thiamine above 1 μg./10 ml. the vitamin B₆ requirement for growth remains essentially constant. Since these higher concentrations of thiamine have been used in methods that utilize this organism for determination of vitamin B₆ (1,2), the validity of these methods is confirmed.In the presence of thiamine, growth was also permitted by additions of the thiamine antagonist, neopyrithiamine. In this case, however, the relationship was fully competitive; i.e., the amount of neopyrithiamine required for growth increased regularly with the thiamine concentration. At concentrations considerably higher than those required for growth, neopyrithiamine again inhibited growth, and this inhibition was prevented by an increase in the thiamine concentration. Thus neopyrithiamine acts by lowering the effective thiamine concentration to subinhibitory levels; if excessive amounts are used, it prevents essential metabolic functions of thiamine and itself becomes toxic. The mechanism by which vitamin B₆ prevents thiamine toxicity is not known.The appearance of a requirement for certain growth factors because of inhibitory effects of other metabolically important compounds, rather than because of an intrinsic inability of the organism to synthesize the growth factor, may be much more common than the few recorded instances of this phenomenon indicate.     

Expression,purification, and activity of ActhiS,a thiazole biosynthesis enzyme from <Emphasis Type="Italic">Acremonium chrysogenum</Emphasis>

Thiamine pyrophosphate is an essential coenzyme in all organisms. Its biosynthesis involves independent syntheses of the precursors, pyrimidine and thiazole, which are then coupled. In our previous study with overexpressed and silent mutants of ActhiS (thiazole biosynthesis enzyme from Acremonium chrysogenum), we found that the enzyme level correlated with intracellular thiamine content in A. chrysogenum. However, the exact structure and function of ActhiS remain unclear. In this study, the enzyme-bound ligand was characterized as the ADP adduct of 5-(2-hydroxyethyl)-4-methylthia-zole-2-carboxylic acid (ADT) using HPLC and ¹H NMR. The ligand-free ActhiS expressed in M9 minimal medium catalyzed conversion of NAD⁺ and glycine to ADT in the presence of iron. Furthermore, the C217 residue was identified as the sulfur donor for the thiazole moiety. These observations confirm that ActhiS is a thiazole biosynthesis enzyme in A. chrysogenum, and it serves as a sulfur source for the thiazole moiety.     

Tritrichomonas foetus: Partly defined cultivation medium for study of the purine and pyrimidine metabolism

A partly defined medium was successfully designed for the cultivation of Tritrichomonas foetus, an anaerobic protozoan parasite of cattle. The medium consists of hypoxanthine, uracil, and thymidine as the sole precursors of nucleotides in T. foetus. Elimination of any one of the three precursors from the medium led to cessation of T. foetus growth. The information provided by this medium verifies our previous observations that T. foetus is incapable of de novo purine and pyrimidine synthesis, that hypoxanthine can be converted to AMP and GMP, that uracil is incorporated into all pyrimidine ribonucleotides including UDP-glucose—the precursor of glycogen synthesis, and that thymidine is the only precursor of TMP. The omission of folate from the medium, without affecting growth of T. foetus, also supports our previous finding that the parasite does not have functioning dihydrofolate reductase or thymidylate synthetase. The successful plating of T. foetus on agar plates incorporating the partly defined medium with near 100% plating efficiency makes it possible to isolate T. foetus mutants for further studies of purine and pyrimidine metabolism in this parasite.