Review
Thiamin metabolism and thiamin diphosphate-dependent enzymes in the yeast Saccharomyces cerevisiae: genetic regulation

https://doi.org/10.1016/S0167-4838(98)00069-7Get rights and content

Abstract

The yeast Saccharomyces cerevisiae utilises external thiamin for the production of thiamin diphosphate (ThDP) or can synthesise the cofactor itself. Prior to uptake into the cell thiamin phosphates are first hydrolysed and thiamin is taken up as free vitamin which is then pyrophosphorylated by a pyrophosphokinase. Synthesis of ThDP starts with the production of hydroxyethylthiazole and hydroxymethylpyrimidine. Those are linked to yield thiamin phosphate which is hydrolysed to thiamin and subsequently pyrophosphorylated. The THI genes encoding the enzymes of these final steps of ThDP production and of thiamin utilisation have been identified. Their expression is controlled by the level of thiamin and a number of regulatory proteins involved in regulated expression of the THI genes are known. However, the molecular details of the regulatory circuits need to be deciphered. Since the nucleotide sequence of the entire yeast genome is known we can predict the number of ThDP-dependent enzymes in S. cerevisiae. Eleven such proteins have been found: pyruvate decarboxylase (Pdc, three isoforms), acetolactate synthase, a putative α-ketoisocaproate decarboxylase with a regulatory role in ThDP synthesis and two proteins of unknown function form the group of Pdc related enzymes. In addition there are two isoforms for transketolase as well as the E1 subunits of pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase. Expression of most of these genes is either induced or repressed by glucose. Surprisingly, it has been found recently that expression of one of the genes for Pdc is repressed by thiamin. In addition, the regulatory protein Pdc2p was shown to be required for high level expression of both the THI and the PDC genes. Apparently, the production of ThDP and of the enzymes using this cofactor is coordinately regulated. Future research will focus on the elucidation of the molecular mechanisms of this novel type of regulation.

Introduction

The yeast Saccharomyces cerevisiae (baker’s or brewer’s yeast) is a unicellular fungal microorganism with a characteristic metabolic activity, namely the fermentation of sugar to ethanol and CO2. Over the years it has served as a convenient source for the isolation of thiamin, vitamin B1, and many enzymes including those dependent on thiamin diphosphate (ThDP). It has also become a prominent model eukaryotic organism and genetic, biochemical and cell biological research on S. cerevisiae has reached a high level of sophistication both in terms of available methods and of the knowledge obtained. This culminated recently in the determination of the nucleotide sequence of the complete genome of a haploid laboratory yeast strain, a total of 12 500 kb on 16 independent chromosomes encoding just over 6000 genes [1].

Like plants and many other microorganisms S. cerevisiae produces ThDP de novo; the vitamin was first isolated from yeast as long ago as 1932 and the structure elucidated in 1936 [2, 3]. However, yeast can also utilise externally provided thiamin as a source for ThDP synthesis, thereby offering the possibility to analyse the ThDP biosynthetic pathway at the genetic level and to study its regulation. In addition, the availability of the complete genome sequence, in combination with genetic and biochemical analyses, allows for the first time the identification and characterisation of the full complement of ThDP-dependent enzymes in a eukaryotic organism. In this review we will first address the present knowledge of yeast ThDP production and then discuss the genetics of ThDP-dependent enzymes. Where relevant, results obtained from studies with S. cerevisiae are compared and contrasted to data from other organisms, especially from fission yeast Schizosaccharomyces pombe. We emphasise the regulation of thiamin metabolism and ThDP-dependent enzymes, at both the transcriptional and post-transcriptional levels, and we point out the preliminary but exciting perspective for an integrated regulation of the production of ThDP and ThDP-dependent enzymes.

Section snippets

Thiamin metabolism

Despite this long history of knowledge and recognition as an essential metabolic cofactor many genetic facets of thiamin biosynthesis and metabolism remain unresolved. This is especially surprising since thiamin auxotrophs were amongst the earliest nutritional mutants isolated from a variety of organisms, including bacteria, fungi [4] and plants [5].

Thiamin diphosphate-dependent enzymes

The conservation of binding sites for ThDP in enzymes using this cofactor as well as the availability of the complete genome sequence of the yeast S. cerevisiae allows the prediction of the full set of ThDP-dependent enzymes in this eukaryotic organism (Table 2). In fact, most but not all of these genes had been identified by genetic analysis before the sequence of the yeast genome became available.

There are five known ThDP-requiring enzymes in yeast that can be divided into three groups.

Group

Regulatory coupling between thiamin metabolism and PDC gene expression

The unexpected discovery that Pdc2p is a positive regulator of the expression of both the THI and the PDC genes has opened a new field of investigation around the central question of whether there is a coupling between the control of thiamin metabolism and the production of ThDP-dependent enzymes. Apparently, the answer is ‘yes’, at least with respect to Pdc. Using a combination of Pdc assays, Northern blots and promoter-LacZ fusions we have established that expression of PDC5, but not of PDC1,

Acknowledgements

We are grateful to Dick Dickinson, Kazuto Nosaka and Charles Singleton, who have informed us about their recent works and kindly allowed us to cite unpublished results. It is also a pleasure to acknowledge the postdoctoral workers and students who have carried out the research reported from our own laboratories, especially Håkan Cederberg, Ines Eberhardt and Elize Muller (SH lab) as well as Robert Burrows, Kerry Byrne, Richard Hather, Louise Kew, Uta Praekelt, Emma Richards and Dave Walsh (PM

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