Chimeric human mitochondrial PheRS exhibits editing activity to discriminate nonprotein amino acids

Abstract

Mitochondria are considered as the primary source of reactive oxygen species (ROS) in nearly all eukaryotic cells during respiration. The harmful effects of these compounds range from direct neurotoxicity to incorporation into proteins producing aberrant molecules with multiple physiological problems. Phenylalanine exposure to ROS produces multiple oxidized isomers: tyrosine, Levodopa, ortho-Tyr, meta-Tyr (m-Tyr), and so on. Cytosolic phenylalanyl-tRNA synthetase (PheRS) exerts control over the translation accuracy, hydrolyzing misacylated products, while monomeric mitochondrial PheRS lacks the editing activity. Recently we showed that "teamwork" of cytosolic and mitochondrial PheRSs cannot prevent incorporation of m-Tyr and l-Dopa into proteins. Here, we present human mitochondrial chimeric PheRS with implanted editing module taken from EcPheRS. The monomeric mitochondrial chimera possesses editing activity, while in bacterial and cytosolic PheRSs this type of activity was detected for the (αβ)2 architecture only. The fusion protein catalyzes aminoacylation of tRNA(Phe) with cognate phenylalanine and effectively hydrolyzes the noncognate aminoacyl-tRNAs: Tyr-tRNA(Phe) and m-Tyr-tRNA(Phe) .

Keywords: ROS-damaged amino acid; aminoacyl-tRNA synthetases; aminoacylation; chimera; editing; fusion protein.

Figure 1

Editing activity of EMD fragments of EcPheRS against Tyr‐tRNA^Phe: (A) B1–B5 and (B) B1–B4. Reactions were performed with 1.2 µM Tyr‐tRNA^Phe prepared from in vitro transcribed E. coli tRNA^Phe, with the addition of 4 µM B1–B5, 4 µM B1–B4, and 1.2 µM chimera or in the absence of enzyme.

Figure 2

(A) “Closed” and (B) “Open” conformations of HmstPheRS. The “Open” conformation is depicted in complex with tRNA^Phe (PDB codes: 3MCQ and 3TUP accordingly). CAM colored green, ABD colored yellow, while tRNA complexed with “Open” state of HmstPheRS colored brown.

Figure 3

Overview of assembly process for human mitochondrial chimeric PheRS. (A) Structural module EMD from EcPheRS. Components of the module–structural domains B1–B5 depicted in different colors and marked accordingly. (B) Overall structure of the HsmtPheRS enzyme in “closed” inactive conformation. (C) The 3D‐model of human mitochondrial chimeric PheRS ribbon representation. (D) The 3D‐model space‐filling representation.

Figure 4

Interface area in the “closed” inactive configuration of HsmtPheRS. CAM is colored blue, while ABD is colored green. Long stretch of aa (11 aa) connecting CAM and ABD colored red. Interface area is outlined by oval colored grey.

Figure 5

Aminoacylation and editing activities of chimera protein in comparison with various wild‐type PheRSs: (A) Charging of E. coli tRNA^Phe transcript (0.8 μM) with Phe by chimera (0.4 µM) or HsmtPheRS (0.4 µM). Reactions were performed at 37°C in the presence of 5 mM ATP and 4 μM [³H]Phe; (B) Editing activities of chimera (1 µM) and EcPheRS (20 nM) toward exogenous Tyr‐tRNA^Phe (1.2 μM); (C) Reaminoacylation of m‐Tyr‐tRNA^Phe with Phe by chimera or HsmtPheRS. The E. coli tRNA^Phe transcript was preaminoacylated with m‐Tyr and purified, then incubated (at 1.2 µM concentration) in the presence of ATP, [³H]Phe, and chimera (0.4 μM) or HsmtPheRS (0.4 µM).