Biosynthesis of vitamin B(6) in yeast: incorporation pattern of glucose.

Two yeasts, Saccharomyces cerevisiae ATCC 7752 and Candida utilis ATCC 9256, were incubated in the presence of variously multiply (13)C-labeled samples of D-glucose. The (13)C incorporation pattern within pyridoxamine dihydrochloride, established by (13)C NMR spectroscopy, differed from that which had previously been found within pyridoxine, isolated from Escherichia coli. Thus, the origin of the carbon skeleton of vitamin B(6) in yeast differs substantially from its origin in E. coli. In particular, in yeast the distribution of (13)C within the C(5) chain C-2',2,3,4,4' of pyridoxamine corresponds to the distribution of (13)C within the C(5) chain C-1,2,3,4,5 of the ribose component of cytidine. It follows that the C(5) chains of pyridoxamine and of ribose originate from a common glucose-derived pentulose or pentose intermediate. By contrast, in E. coli the C(5) chain of pyridoxine is derived from 1-deoxy-D-xylulose 5-phosphate which, in turn, originates by condensation of pyruvic acid with glyceraldehyde 3-phosphate.

The biogenetic anatomy of vitamin B6. A 13C NMR investigation of the biosynthesis of pyridoxol in Escherichia coli.

It is shown by incorporation experiments with 13C bond-labeled substrates, followed by analysis by means of 13C NMR spectroscopy, that two compounds, 1-deoxy-D-xylulose (12) and 4-hydroxy-L-threonine (), serve as precursors of pyridoxol (vitamin B6) (1) in Escherichia coli. Together, these two compounds account for the entire C8N skeleton of the vitamin. 1-Deoxy-D-xylulose supplies the intact C5 unit, C-2',2,3,4,4' of pyridoxol. 4-Hydroxy-L-threonine undergoes decarboxylation in supplying the intact C3N unit, N-1,C-6,5,5'. Both precursors are ultimately derived from glucose. The C5 unit of pyridoxol that is derived from 1-deoxy-D-xylulose originates by union of a triose phosphate (yielding C-3,4,4') with pyruvic acid (which decarboxylates to yield C-2',2). D-Erythroate (11) enters the C3 unit, C-6,5,5', and is therefore an intermediate on the route from glucose into 4-hydroxy-L-threonine.

Growth response to 4-hydroxy-L-threonine of Escherichia coli mutants blocked in vitamin B6 biosynthesis.

Mutants of Escherichia coli (pdx B and pdx C) which are blocked in the biosynthesis of pyridoxol (vitamin B6) showed a growth response to 4-hydroxy-L-threonine. This observation constitutes the first direct evidence in support of the view that 4-hydroxy-L-threonine is implicated in the biosynthesis of vitamin B6. 1-Aminopropan-2,3-diol, the decarboxylation product of 4-hydroxy-L-threonine, does not support the growth of these mutants. Deuterium from deuterium-labelled 1-aminopropan-2,3-diol was not incorporated into pyridoxol.

The regiochemistry and stereochemistry of the biosynthesis of vitamin B6 from triose units.

13C and 2H NMR spectroscopy has been employed to probe the biosynthesis of vitamin B6 in Escherichia coli. The 13C NMR spectrum of a sample of pyridoxol derived biosynthetically from D-[1,2,3,4,5,6-13C6]glucose shows that the bonds, C(2)-C(3) and C(4)-C(5), of the pyridine nucleus are the only two carbon-carbon bonds of pyridoxol which are generated de novo in the course of its biosynthesis from glucose. It follows that the pyridoxol skeleton is generated from two intact triose units and a triose-derived two-carbon unit, all of which are supplied by glucose. From the 2H NMR spectra of samples of pyridoxol derived from (R)-[1,1-2H2]glycerol and (S)-[1,1-2H2]glycerol, respectively, it can be deduced that the rehydroxymethyl group of glycerol enters C-2', C-4', and C-5' of the pyridoxol skeleton. It follows that each of the three fragments is derived from glycerol in stereo-specific fashion. These results answer questions concerning the regiochemistry and the stereochemistry of pyridoxol biosynthesis.

The status of glycolaldehyde in the biosynthesis of vitamin B6.

Competition experiments, employing 14C-labeled samples of glycerol and glycolaldehyde, indicate that in Escherichia coli B there are two independent pathways leading to pyridoxal. In mutant WG2 (and therefore presumably also in the wild strain) the major pathway utilizes glycerol and related trioses as the sole carbon source in the construction of the C8N skeleton of pyridoxol: C-1, -3 of glycerol yields C-2', -3, -4', -5' and -6, C-2 of glycerol yields C-2, -4, and -5 of the vitamin. In the minor pathway glycolaldehyde and not glycerol supplies C-5 and C-5' of pyridoxol, while glycerol is the source of the other 6 carbon atoms. In mutant WG3 the major route is blocked and the "glycolaldehyde pathway" becomes the sole source of vitamin B6.

Thiamin biosynthesis in Saccharomyces cerevisiae. Origin of carbon-2 of the thiazole moiety.

Radioactivity from [2-14C]glycine enters C-2 of the thiazole moiety of thiamin and no other site, in Saccharomyces cerevisiae (strains A.T.C.C. 24903 and 39916, H.J. Bunker). Radioactivity from L-[Me-14C]methionine or from DL-[2-14C]tyrosine does not enter thiamin.

The biosynthetic route from ornithine to proline.

It is shown by tracer experiments with DL-[2-3H,5-14C]- and DL-[(RS)-5-3H,5-14C]rnithine, that the metabolic conversion of ornithine into proline, in three plant species (Nicotiana tabacum, Datura stramonium, and Lupinus angustifolius), takes place with maintenance of the delta-hydrogen atoms but with loss of the alpha-hydrogen atom. This indicates a route via alpha-keto-delta-aminovaleric acid (5-amino-2-oxopentanoic acid) and disproves the accepted route via glutamic gamma-semialdehyde (2-amino-5-oxopentanoic acid).

Biosynthesis of vitamin B6: incorporation of three-carbon units.

Pyridoxol, one of the forms of vitamin B(6), is derived from three glycerol units. One of these is incorporated by way of pyruvate as a two-carbon fragment at the oxidation level of acetaldehyde. The other two glycerol units are incorporated intact, possibly by way of triose phosphate.