Biosynthesis of the thiazole moiety of thiamin (vitamin B1) in higher plant chloroplasts.

Abstract

The chloroplast stromal proteins from spinach condense the two heterocyclic moieties of vitamin B,-4-methyl-5-(J3-hydroxyethyl)thiazole and 2-methyl-4-amino-5-hydroxymethylpyrimidine-in the presence of MgATP. We have taken advantage of this observation to study thiazole synthesis. We present data indicating that pyruvate, glyceraldehyde 3-phosphate,-tyrosine, cysteine, and MgATP are required for this synthesis and that pyruvate and glyceraldehyde 3-phosphate can be replaced by 1-deoxy-D-threo-2-pentulose. The thiazole synthesis occurs at a sustained, low rate of 140 pmol per mg of protein per hr.It is remarkable that in 1990, more than half a century after the discovery and synthesis of vitamin B1 (1), the early biosynthetic steps of this important molecule remain obscure. The pathways have been studied by feeding microorganisms with small quantities of labeled precursors and accurately determinating the distribution of incorporated label. Nevertheless, the enzymes involved in the biosynthesis of the cyclic moieties of vitamin B1-2-methyl-4-amino-5-hydroxymethylpyrimidine and 4-methyl-5-(q-hydroxyethyl)thiazole-are still unknown.A 1-deoxy-D-threo-2-pentulose probably produced by the condensation of pyruvate and a triose phosphate has been proven to be the precursor of the contiguous five-carbon unit (C4'-C4-C5-C5'-C5") of the thiazole ring (refs. 2-4; however, see ref. 5), while the C2-N3 unit of this ring derives from the adjacent carbon and nitrogen atoms of glycine in Saccharomyces cerevisiae (6-8) or of tyrosine in Escherichia coli (9-11) and sulfur comes from cysteine (12)(13)(14). The pyrimidine moiety of vitamin B1 has been postulated to be derived from 5'-phosphoribosyl-5-aminoimidazole (15,16). The pyruvate dehydrogenase complex has been reported to be involved in the condensation of pyruvate and glyceraldehyde 3-phosphate (17, 18) to produce 1-deoxy-D-threo-2-pentulose.In higher plants, many enzyme-coenzyme complexes are unstable during purification. For example, highly purified preparations of pyruvate dehydrogenase from plant mitochondria are completely dependent on added thiamin pyrophosphate for enzymatic activity, but similar preparations of the mammalian enzymes show little or no response to exogenous thiamin pyrophosphate (19). If enzyme-coenzyme complexes are also unstable in vivo, the synthesis of vitamin B1 should be continuous and a significant rate of synthesis should be observed. Indirect evidence from mutants ofPisum sativum L. with altered pyrimidine moiety synthesis (20) shows that a high level of exogenous thiamin is required in order to restore normal cellular metabolism. Nevertheless, the biosynthetic pathways of the pyrimidine and thiazole rings of vitamin B1 in higher plants are totally unexplored.Here, we report evidence that the chloroplast is a site for the synthesis of the thiazole moiety of vitamin B1, which derives from 1-deoxy-D-threo-2-pentulose, tyrosine, and cysteine. MATERIALS AND METHODS Preparation of Soluble (Stromal) Proteins from Spinach LeafChloroplasts. Al...

AtTHIC, a gene involved in thiamine biosynthesis in Arabidopsis thaliana

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Dedicated Roles of Plastid Transketolases during the Early Onset of Isoprenoid Biogenesis in Pepper Fruits1

“…This pathway has no steps in common with cytoplasmic IPP synthesis, which proceeds via the mevalonic acid pathway. Our data also suggest that plastid isoprenoid biosynthesis is tightly coupled to that of thiamine (vitamin B 1 ) (David et al., 1981;Julliard and Douce, 1991) and pyridoxine (vitamin B 6 ) (Hill et al, 1989;Julliard, 1992) biosynthesis. …”

Overexpression of Plastid Transketolase in Tobacco Results in a Thiamine Auxotrophic Phenotype

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