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Chapter 24 : Amino Acids and Polyamines: Polyfunctional Proteins, Metabolic Cycles, and Compartmentation
Category: Microbial Genetics and Molecular Biology; Fungi and Fungal Pathogenesis
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This chapter focuses on three unusual aspects of certain of these pathways: polyfunctional proteins, metabolic cycles, and compartmentation. The study of aromatic amino acid biosynthesis in Neurospora crassa provided some of the earliest insights into unusual genetic relationships among related metabolic enzymes. Most fungi, including yeasts, display a global response to amino acid deprivation. In N. crassa and other filamentous fungi, it is referred to as cross-pathway control, with many of the genes carrying the cpc designation. In S. cerevisiae, the system is called general amino acid control, with gene names starting with GC. It is referred to here as cross-pathway control and is described briefly to fill out the physiological aspects of amino acid synthesis and control. Polyamine metabolism in fungi has been studied mainly in S. cerevisiae and N. crassa. The chapter focuses on features of fungi that organize and optimize metabolic systems. The management of polyamine pools has revealed a complex physiological landscape in which enzyme regulation, metabolic flow, small-molecule binding, and vacuolar activity converge. Very little catabolism of polyamines takes place in N. crassa, even at high external amine concentrations. The ionic binding of spermidine and basic amino acids to polyphosphate in the vacuole explains why the intact organelle becomes so dense upon isolation in sucrose gradients, where they lose water as they sediment. Indeed, it is likely that in fungi, as in plants, they have important roles in fine-tuning and otherwise managing metabolic and ionic traffic, varying widely with the species and the environment.
Pathway of chorismate synthesis in N. crassa (top), showing the metabolic positions of aro mutations. The organization of the ARO gene complex on linkage group IIR (bottom) shows the order of the catalytic domains (1 through 5) and of the corresponding component elements of the complex (aro-1, -2, -4, -5, and -9). The complementation groups are represented by lines that group mutants that will not complement; lines that do not overlap signify that complementation will take place between members of the different groups. Note that the translational polarity in this figure is oriented right (N terminal) to left (C terminal). Redrawn from Rines et al., 1969 , and reprinted from Davis, 2000 .
Tryptophan, phenylalanine, and tyrosine synthesis in N. crassa, showing the metabolic positions of various genes. Note that the complex of trp-1 and trp-2 gene products catalyzes three nonsequential reactions. Not all substrates are shown; for details of the tryptophan synthase reaction, see Fig. 3 . Reprinted from Davis, 2000 .
The tryptophan synthase partial reactions at sites I (reaction 2) and II (reaction 3). The intermediate indole diffuses between sites I and II, largely confined within the protein. Reprinted from Davis, 2000 .
Isoleucine, valine, and leucine biosynthesis in N. crassa, showing the metabolic positions of mutations. Note that the ilv-3 (acetohydroxy acid synthase), ilv-2 (reductoisomerase), and ilv-1 (dehydratase) genes encode single enzymes that carry out reactions in both the isoleucine and valine pathways. Reprinted from Davis, 2000 .
The pathways of pyrimidine, arginine, proline, and polyamine synthesis in N. crassa, showing the localization of enzymes and the metabolic positions of mutations. See Table 1 for gene-enzyme assignments. Reprinted from Davis, 2000 .
Carbamyl phosphate (CAP) overflow between pathways in two types of double mutants of Neurospora. Mutations pyr-3d (top) and arg-12 s (bottom) block CAP utilization in one pathway and thereby relieve nutritional requirements imposed by CPS mutants in the other. Parenthesized compounds are not synthesized. CAP is arginine or pyrimidine specific. Reprinted from Davis, 1972 .
Steady-state flux of arginine and ornithine in exponential cultures of N. crassa grown in minimal medium. Values are in nanomoles per minute per milligram, dry weight, of mycelium. Boxed numbers identify ornithine decarboxylase (1), ornithine transaminase (2), and ornithine carbamoyltransferase (3). Based on Karlin et al., 1976 , and reprinted from Davis, 2000 .
Polyamine synthesis in N. crassa, with the structures of relevant intermediates and the metabolic positions of mutations. The dotted line signifies allosteric feedback inhibition of argi-nine synthesis by arginine. MTA, methylthioadenosine. Reprinted from Davis, 2000 .
Relationship between spermidine concentrations within and outside mycelial cells growing in the presence of different concentrations of spermidine. The strain used carries an ODC-deficient spe-1 mutation; therefore, all spermidine in the cells originates in the medium. Cellular spermidine is expressed as nanomoles of spermidine per milligram, dry weight (left ordinate) or as millimolar in cell water (2.5 ml per g, dry weight) (right ordinate). The dotted line shows the 1:1 relationship expected of a diffusional equilibrium. Over the range from 0.5 to 5 mM spermidine in the medium, cellular spermi-dine rises from 9 to ~14 mM (solid line). The arrow on the left ordinate shows the normal concentration of spermidine in wild-type cells grown in minimal medium. Reprinted from Davis, 2000 .
Gene-enzyme assignments in the arginine, proline, pyrimidine, and polyamine pathways