Pyramin

Toxopyrimidine (pyramin lOlO) may be obtained from thiamine (vitamin Bi 1011) by acidic hydrolysis or by treatment with the thiaminase of Bacillus aneurinolyticus. Toxopyrimidine produces convulsions and death in rodents as do analogues, e.g. 2,4-dimethylpyrimidin-5-ylmethanol (1012) (65JMC750), but the effect is minimized or even... 

V. 5-Amino-l-((3-D-ribofuranosyl)imidazole 5 -Phosphate, the Precursor of Pyramine in... 

Still more confusion plagued early researches, when it was not realized that the biosynthetic routes to thiamine in prokaryotes and eukaryotes are quite different, a fact not expected at the outset. Thus, evidence collected from the study of yeast could not be transposed to bacteria, and vice-versa. For instance, formate is a most efficient precursor of one of the carbon atoms of the pyrimidine part of thiamine (pyramine), both in yeasts and enterobacteria, but incorporates at C-2 in bacteria and at C-4 in yeast. However, as is briefly covered in Section VIII, this dichotomy of pathways might have a deep significance in the perspective of biochemical evolution during primitive life on Earth. 

Thiamine is present in cells as the free form 1, as the diphosphate 2, and as the diphosphate of the hydroxyethyl derivative 3 (Scheme 1) in variable ratio. The component heterocyclic moieties, 4-amino-5-hydroxymethyl-2-methylpyrimidine (4) and 4-methyl-5-(2-hydroxyethyl)thiazole (5) are also presented in Scheme 1, with the atom numbering. This numbering follows the rules of nomenclature of heterocyclic compounds for the ring atoms, and is arbitrary for the substituents. To avoid the use of acronyms, compound 5 is termed as the thiazole of thiamine or more simply the thiazole. This does not raise any ambiguity because unsubstituted thiazole is encountered in this chapter. Other thiazoles are named after the rules of heterocyclic nomenclature. Pyrimidine 4 is called pyramine, a well established name in the field. A detailed account of the present status of knowledge on the biosynthesis of thiamine diphosphate from its heterocyclic moieties can be found in a review by the authors.1 This report provides only the minimal information necessary for understanding the main part of this chapter (Scheme 2). 

Thiamine can be considered to be the product of the quatemization of 4-methyl-5-(2-hydroxymethyl)thiazole (5) by an active derivative of 4-amino-5-(hydroxymethyl)-2-methyl pyrimidine (4) (Scheme 2). In living cells, pyramine can be activated by conversion into the diphosphate 7, via monophosphate 6, and the substrate of the enzyme responsible for the quatemization is not the thiamine thiazole, but its phosphate 8. The product of the condensation, thiamine phosphate (9), is finally converted into diphosphate 2—the biochemically active derivative—by hydrolysis to free thiamine, followed by diphosphorylation, or more directly, in some cases. Enzymes are known for all of the steps depicted in Scheme 2, and adenosine triphosphate (ATP) is, as usual, the phosphate donor. 

Now a cautionary note is necessary. This chapter covers with the biosyntheses of thiazole and pyramine. From a glance at Scheme 2, one might conclude that... 

Scheme 7.—The hydrolytic ring opening of pyramine, producing fragments in which the specific activity of each carbon can be estimated.

An extract from the soluble stromal proteins of purified and intact spinach-leaf chloroplasts was prepared by lysis of the cells in buffer, centrifugation of the suspension of broken cells, and concentration of the supernatant with removal of insoluble material. This extract contained all of the enzymes involved in the condensation of the cyclic moieties of thiamine, thiazole, and pyramine. Thus, the synthesis of thiamine in this extract following the addition of pyramine and putative precursors was a proof that the system had the possibility of building the thiazole. It was found that L-tyrosine was the donor of the C-2 carbon atom of thiazole, as in E. coli. Also, as in E. coli cells, addition of 1 -deoxy-D-f/irco-pen-tulose permitted synthesis of the thiamine structure. The relevant enzymes were localized by gel filtration in a fraction covering the 50- to 350-kDa molecular-mass range. This fraction was able to catalyze the formation of the thiazole moiety of thiamine from 0.1 -mM 1-deoxy-D-t/ireo-pentulose at the rate of 220 pmol per mg of protein per hour, in the presence of ATP and Mg2+. 

From this observation of the inhibition by adenosine, and other observations, Newell and Tucker suspected the existence of a common synthetic pathway for adenosine and thiamine, and proved (with the help of a collection of mutants) that the bifurcation occurred after the 5-amino- l-(P-D-ribofura-nosyl)imidazole 5 -phosphate (46) step (Scheme 23). Finally, they found that 5-amino-l-(0-D-ribofuranosyl)imidazole (47), labeled with l4C in the imidazole ring, was incorporated into pyramine without significant loss of molar radioactivity by a mutant that is able to use this nucleoside (presumably after phosphorylation).53,54... 

Scheme 23.—Some important steps in the biosynthesis of adenosine 5 -phosphate, and the branching of the sequence at the AIR level, leading ultimately to pyramine, in S. ryphimurium cells.

Scheme 24.—Correspondence between the carbon atoms of AIR and those of pyramine, and the mode of opening of the imidazole ring in the ring expansion in the synthesis of pyramine.

The experiments with (U-l3C)AIRs showed that this nucleoside supplied all of the carbon atoms of pyramine. Because out of 6 carbon atoms of pyramine, only three may come from the imidazole part of AIRs, it can be concluded that the three other carbon atoms come from the ribose part of this nucleoside. In complete agreement with these results, radioactivity from AIRs, labeled mainly with, 4C in its ribose part, was found to incorporate into the three carbon atoms of pyramine, the origin of which was, at the time, unknown. Owing to the minute amount of AIRs supplied (as compared with that of glucose) in both experiments, the incorporation of label from AIRs after metabolic degradation is ruled out. 

Further experiments with labeled precursors were necessary to shed a little more light on this puzzling observation. Pyramine, biosynthesized from AIRs labeled with, 4C on C-l on the ribose part, exhibited only marginal radioactivity. This result rules out C-l of ribose in AIRs as a precursor of pyramine. This conclusion was confirmed with a precursor labeled at the C-l position with the stable l3C isotope. The mass spectrum of the ethylthio derivative of pyramine was identical with that of an unlabeled sample (Scheme 9). 

On the other hand, the fragmentation of pyramine obtained from (2 -l3C)AIRs indicated clearly that C-2, in the ribose part, was the precursor of carbon C-7 of the methyl on C-2 of the pyrimidine ring (Scheme 29). This result was confirmed by an experiment with a sample of AIRs labeled with l4C on C-l, C-2, C-3, on the ribose, and C-5 on the imidazole, with an approximate distribution of 1, 1, 3, 3. This precursor produced pyramine with the methyl group almost as radioactive as C-l or C-2, and much less than C-3 of AIRs. Because of the incorporation of C-5 of imidazole into C-4 of pyramine, and the comparable activities of C-3 and C-5 in the precursor AIRs, the specific activity of pyramine... 

Scheme 29.—Carbon atom C-2 of AIRs is the precursor of the methyl of pyramine. The labeled atoms are printed in bold type.

Scheme 30.—Correspondence between ribose and pyramine carbon atoms in the biosynthesis by S. typhimurium.

Scheme 31.—Correspondence between the nitrogen atoms of AIRs and those of pyramine in S. typhimurium.

It has been shown already that C-2 of ribose is the precursor of the methyl group, and C-l is eliminated in the biosynthesis. The following observation can be pertinent to the point. Pyrimidine (58) is very unstable and quickly decar-boxylates in aqueous solution at room temperature to give pyramine (Scheme 32).67 Thus, if a C-l -C-2 fragment of the ribose part of AIRs became attached by C-2 to C-2 of a pyrimidine, oxidation of C-l to produce a carboxylic acid function could result in its smooth elimination. 

Much less is known about the participation of sugars in the biosynthesis of pyramine in yeasts, and although it has been proven that sugars can provide some carbon atoms, the exact nature of the more advanced intermediates of sugar origin is not yet clear. Some features of the biosynthesis in S. cerevisiae are summarized in Scheme 33. Two l5N atoms from DL-(l,3-,5N2)histidine were incorporated into the N-3 and amino nitrogen atoms of pyramine. The nitrogen atom of (,5N)aspartate, a known precursor of N-l of histidine, was incorporated into pyramine without dilution.58-70 It was also found that N-l and C-2 of pyramine came respectively from N-l and C-2 of pyridoxol.71-73... 

Scheme 33.—The participation of L-histidine and pyridoxol to the biosynthesis of pyramine yeast.

Scheme 34.—The participation of D-glucose to the biosynthesis of pyramine by C. urilis.

VII, The Distribution of the Four Biosynthetic Routes in Nature 1. Biosynthesis of Pyramine... 

Escherichia coli Adenine and adenosine are inhibitory74 and the synthesis of thiamine can be derepressed by culture in their presence.13,75 adth- Mutants are known.76 [l4C]Formate incorporates at C-2 of pyramine without dilution of molar activity. Glycine labeled with stable isotopes was fed to E. coli and the pyramine was analyzed by mass spectrometry. The two carbon atoms of glycine separated during the biosynthesis. The carboxyl was found12 at C-4, and the C-N fragment was the precursor of C-6-N-1. In conclusion, it is beyond doubt that pyramine synthesis follows the AIR pathway in E. coli. 

Enterobacterbacter aerogenes adth Mutants have been isolated,77 and adenine inhibits the synthesis of thiamine.74,77 [l4C]Formate incorporates at C-2 of pyramine.78... 

Micrococcus denitrificans Adenosine derepresses the enzymes involved in the coupling of pyramine with thiazole.79... 

Spinach chloroplasts There is some evidence that pyramine originates from AIR.80... 

Although the gross features of the biosyntheses of thiazole and pyramine have been elucidated, nothing is known about the nature and order of the individual steps. The relevant enzymes have not yet been found, although it might be hoped that the knowledge accumulated on the precursors and the paths of atoms will help in this respect. An attempt has been made recently to find the genes involved in the biosynthesis of thiazole.82... 

Potassium native gellan, 389-391,430-431 Pseudo-oligosaccharides, spirodioxanyl, 220-221 Pyramine... 

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