Sulfur amino acid biosynthesis in Saccharomyces cerevisiae involves a large number of enzymes required for the de novo biosynthesis of methionine and cysteine and the recycling of organic sulfur metabolites. This review summarizes the details of these processes and analyzes the molecular data which have been acquired in this metabolic area. Sulfur biochemistry appears not to be unique through terrestrial life, and S. cerevisiae is one of the species of sulfate-assimilatory organisms possessing a larger set of enzymes for sulfur metabolism. The review also deals with several enzyme deficiencies that lead to a nutritional requirement for organic sulfur, although they do not correspond to defects within the biosynthetic pathway. In S. cerevisiae, the sulfur amino acid biosynthetic pathway is tightly controlled: in response to an increase in the amount of intracellular S-adenosylmethionine (AdoMet), transcription of the coregulated genes is turned off. The second part of the review is devoted to the molecular mechanisms underlying this regulation. The coordinated response to AdoMet requires two cis-acting promoter elements. One centers on the sequence TCACGTG, which also constitutes a component of all S. cerevisiae centromeres. Situated upstream of the sulfur genes, this element is the binding site of a transcription activation complex consisting of a basic helix-loop-helix factor, Cbf1p, and two basic leucine zipper factors, Met4p and Met28p. Molecular studies have unraveled the specific functions for each subunit of the Cbf1p-Met4p-Met28p complex as well as the modalities of its assembly on the DNA. The Cbf1p-Met4p-Met28p complex contains only one transcription activation module, the Met4p subunit. Detailed mutational analysis of Met4p has elucidated its functional organization. In addition to its activation and bZIP domains, Met4p contains two regulatory domains, called the inhibitory region and the auxiliary domain. When the level of intracellular AdoMet increases, the transcription activation function of Met4 is prevented by Met30p, which binds to the Met4 inhibitory region. In addition to the Cbf1p-Met4p-Met28p complex, transcriptional regulation involves two zinc finger-containing proteins, Met31p and Met32p. The AdoMet-mediated control of the sulfur amino acid pathway illustrates the molecular strategies used by eucaryotic cells to couple gene expression to metabolic changes.

Metabolism of sulfur amino acids in Saccharomyces cerevisiae.

In order to enhance the yield and productivity of metabolite production, researchers have focused almost exclusively on enzyme amplification or other modifications of the product pathway. However, overproduction of many metabolites requires significant redirection of flux distributions in the primary metabolism, which may not readily occur following product deregulation because metabolic pathways have evolved to exhibit control architectures that resist flux alterations at branch points. This problem can be addressed through the use of some general concepts of metabolic rigidity, which include a means for identifying and removing rigid branch points within an experimental framework.

https://science.sciencemag.org/content/sci/252/5013/1675.full.pdf

Network rigidity and metabolic engineering in metabolite overproduction.

The two main contributions of this are the solidification of Corynebacterium glutamicum biochemistry guided by bioreaction network analysis, and the determination of bansal metabolic flux distributions during growth and lysine synthesis. Employed methodology makes use of stoichiometrically based mass balances to determine flux distributions in the C. glutamicum metabolic network. Presented are a brief description of the methodology, a through literature review of glutamic acid bacteria biochemistry, and specific results obtained through a combination of fermentation studies and analysis-directed intracellular assays. The latter include the findings of the lack of activity of glyoxylate shunt, and that phosphoenolpyruvate carboxylase (PPC) is the only anaplerotic reaction expressed in C. glutamicum cultivated on glucose minimal media. Network simplifications afforded by the above findings facilitated the determination of metabolic flux distributions under a variety of culture conditions and led to the following conclusions. Both the pentose phosphate pathway and PPC support fluxes during growth and lysine overproduction branch point does not appear to limit lysine synthesis.

Metabolic flux distributions in Corynebacterium glutamicum during growth and lysine overproduction.

In many organisms, metabolite interconversion at the phosphoenolpyruvate (PEP)-pyruvate-oxaloacetate node involves a structurally entangled set of reactions that interconnects the major pathways of carbon metabolism and thus, is responsible for the distribution of the carbon flux among catabolism, anabolism and energy supply of the cell. While sugar catabolism proceeds mainly via oxidative or non-oxidative decarboxylation of pyruvate to acetyl-CoA, anaplerosis and the initial steps of gluconeogenesis are accomplished by C3- (PEP- and/or pyruvate-) carboxylation and C4- (oxaloacetate- and/or malate-) decarboxylation, respectively. In contrast to the relatively uniform central metabolic pathways in bacteria, the set of enzymes at the PEP-pyruvate-oxaloacetate node represents a surprising diversity of reactions. Variable combinations are used in different bacteria and the question of the significance of all these reactions for growth and for biotechnological fermentation processes arises. This review summarizes what is known about the enzymes and the metabolic fluxes at the PEP-pyruvate-oxaloacetate node in bacteria, with a particular focus on the C3-carboxylation and C4-decarboxylation reactions in Escherichia coli, Bacillus subtilis and Corynebacterium glutamicum. We discuss the activities of the enzymes, their regulation and their specific contribution to growth under a given condition or to biotechnological metabolite production. The present knowledge unequivocally reveals the PEP-pyruvate-oxaloacetate nodes of bacteria to be a fascinating target of metabolic engineering in order to achieve optimized metabolite production.

The PEP-pyruvate-oxaloacetate node as the switch point for carbon flux distribution in bacteria.

(1979). The polysaccharides of green, red and brown seaweeds: Their basic structure, biosynthesis and function. British Phycological Journal: Vol. 14, No. 2, pp. 103-117.

https://www.tandfonline.com/doi/pdf/10.1080/00071617900650121

The polysaccharides of green, red and brown seaweeds: Their basic structure, biosynthesis and function

Regulation of the TCA and glyoxylate cycles in Brevibacterium flavum. II. Regulation of phosphoenolpyruvate carboxylase and pyruvate kinase.

Regulation of the TCA and Glyoxylate Cycles in Brevibacterium flavum: I. Inhibition of lsocitrate Lyase and Isocitrate Dehydrogenase by Organic Acids Related to the TCA and Glyoxylate Cycles

Regulation of nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase from Brevibacterium flavum,a glutamate-producing bacterium

Concerted inhibition of isocitrate dehydrogenase by glyoxylate plus oxalacetate.

Out of 27 strains of methionine auxotrophs of Brevibacterium flavum, 14 strains did not grow on homoserine but grew on O-acetylhomoserine, and all were found to lack homoserine O-acetyltransferase [EC 2.3.1.31] alone. Another 3 strains did not grow on O-acetylhomoserine but grew on homocysteine, and the two strains tested were found to lack O-acetylhomoserine sulfhydrylase (AHS) alone, without any changes in the activities of cystathionine gamma-synthase [EC4.2.99.9] and beta-cystathionase [EC 4.4.1.8]. Prototrophic revertants of the AHS-lacking mutants showed concomitant reversion of AHS activity. None of the methionine auxotrophs grew on cystathionine. From these results it was concluded that the methionine biosynthetic pathway of this bacterium involves formation of O-acetylhomoserine from homoserine by the action of homoserine O-acetyltransferase, and direct formation of homocysteine from O-acetylhomoserine by the AHS reaction. AHS synthesis was strongly repressed by methionine. AHS was purified to 70% purity. The purified preparation was activated by pyridoxal phosphate after treatment with hydroxylamine. The enzyme showed a molecular weight of 360,000, an optimum pH of 8.7 for activity, and specifically reacted with O-acetyl-L-homoserine and showed with O-acetyl-L-serine one hundredth as much activity as that with O-acetylo -homoserine, but did not show activity with O-succinyl-L-homoserine, homoserine, or serine. The Km values for O-acetylhomoserine and H2S were 2.0 mM and 0.08 mM, respectively. The enzyme was inhibited 50, 23. and 29% by 10 mM L-methionine, l-homoserine, and O-acetyl-L-serine, respectively, but it was not inhibited by cystathionine or S-adenosyl-L-methionine.

Hachiro Ozaki

Papers

Regulation of the TCA and glyoxylate cycles in Brevibacterium flavum. II. Regulation of phosphoenolpyruvate carboxylase and pyruvate kinase.

Regulation of the TCA and Glyoxylate Cycles in Brevibacterium flavum: I. Inhibition of lsocitrate Lyase and Isocitrate Dehydrogenase by Organic Acids Related to the TCA and Glyoxylate Cycles

Regulation of nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase from Brevibacterium flavum,a glutamate-producing bacterium

Concerted inhibition of isocitrate dehydrogenase by glyoxylate plus oxalacetate.

Methionine biosynthesis in Brevibacterium flavum: properties and essential role of O-acetylhomoserine sulfhydrylase.