Metabolic origin of the fused aminoacyl-tRNA synthetase, glutamyl-prolyl-tRNA synthetase

Abstract

About 1 billion years ago, in a single-celled holozoan ancestor of all animals, a gene fusion of two tRNA synthetases formed the bifunctional enzyme, glutamyl-prolyl-tRNA synthetase (EPRS). We propose here that a confluence of metabolic, biochemical, and environmental factors contributed to the specific fusion of glutamyl- (ERS) and prolyl- (PRS) tRNA synthetases. To test this idea, we developed a mathematical model that centers on the precursor-product relationship of glutamic acid and proline, as well as metabolic constraints on free glutamic acid availability near the time of the fusion event. Our findings indicate that proline content increased in the proteome during the emergence of animals, thereby increasing demand for free proline. Together, these constraints contributed to a marked cellular depletion of glutamic acid and its products, with potentially catastrophic consequences. In response, an ancient organism invented an elegant solution in which genes encoding ERS and PRS fused to form EPRS, forcing coexpression of the two enzymes and preventing lethal dysregulation. The substantial evolutionary advantage of this coregulatory mechanism is evidenced by the persistence of EPRS in nearly all extant animals.

Keywords: EPRS; aminoacyl tRNA synthetase; citric acid cycle; evolution; fusion protein; gene fusion; glutamate; mathematical modeling; molecular evolution; tricarboxylic acid cycle (TCA cycle) (Krebs cycle).

Figure 1.

Origin of EPRS. Fused EPRS appeared in an ancestor C. owczarzaki near the basal root of Metazoa and close to the time of the appearance of the MSC.

Figure 2.

Biosynthetic pathways of amino acids. Ten of the 20 amino acids are derived from two intermediates of the citric acid cycle. The glyoxylate cycle (dashed curve), feedback inhibition of proline synthesis by proline, and inhibition of the citric acid cycle by hypoxia are noted.

Figure 3.

The dynamics of key system outputs in fused and unfused systems. Shown are time courses of glutamic acid (red dashed curve), proline (blue dashed curve), ERS (red solid curve), and PRS (blue solid curve) in the unfused ERS-PRS system (A) and glutamic acid, proline, and EPRS (purple curve) in the fused EPRS system (B). For the simulations, [α-KG] was set at 5; rate constants k₁ to k₅ and k_Esyn, k_Psyn, and k_EPsyn were set at 0.1; k_Edeg, k_Pdeg, and k_EPdeg were set at 0.05; and 0.0001 was used for δ. Initial values of [E], [P], [ERS], [PRS], and [EPRS] were set at 1.

Figure 4.

Effect of proline utilization on system outputs. The effects of increased proline utilization rate on [E], [P], [ERS], [PRS], and [EPRS] were determined at k₅ set to 0.11 (A and B), 0.13 (C and D), and 0.15 (E and F) for the ERS-PRS (A, C, and E) and EPRS (B, D, and F) systems. Other system parameters and outputs were as in Fig. 3.

Figure 5.

Effect of α-KG level on system outputs. The concentration of [α-KG] was set to 5.0 (A and B), 4.5 (C and D), and 4.0 (E and F) for the ERS-PRS (A, C, and E) and EPRS (B, D, and F) systems. Other system parameters and outputs were as in Fig. 3. To permit direct comparison, Fig. 3, A and B, are redisplayed here in A and B.

Figure 6.

Combined influence of increased proline utilization and decreased α-KG on system outputs. The rate of proline utilization, k₅, was set at 0.10, and [α-KG] was set at 5.0 (A and B), or k₅ was set at 0.13, and [α-KG] was set at 4.5 (C and D) for the ERS-PRS (A and C) and EPRS (B and D) systems. Other system parameters and outputs were as in Fig. 3. To permit direct comparison, Fig. 3, A and B, are redisplayed here in A and B.

Figure 7.

Alternate solutions to counter negative effects of increased proline utilization and reduced α-KG. A, k₁, the rate of α-KG conversion to E, was increased from 0.10 to 0.12. B, k₃, the rate of incorporation of E into protein, was decreased from 0.10 to 0.05. C, k_Esyn, the rate of synthesis of ERS, was reduced from 0.1 to 0.05. D, removal of negative regulation of PRS by [P]. Unless stated otherwise, other system parameters and outputs are as in Fig. 3.

Figure 8.

Comparison of three systems in response to altered α-KG concentration. The concentration of α-KG was varied, and the steady-state cellular pool of free proline (A) and free glutamic acid (B) was determined in the ERS-PRS, EPRS, and ERS-ΔPRS systems. Other system parameters are as in Fig. 3.