E. Coli Lysyl-tRNA Synthetase (LysU): Structure And Function Studies

Diadenosine 5',5'"-P1,P4-tetraphosphate (AP4A) is the most common dinucleoside polyphosphate and has been found in various organisms. It is involved in diverse intracellular and extracellular biological roles as "alarmone", such as cell apoptosis, DNA repair and cellular stress responses.E. coli heat-inducible lysyl-tRNA synthetase (LysU) is an efficient Ap4A synthetase with 80% of total AP4A synthesis in E. coli cell extracts. The synthesis of Ap4A catalyzed by LysU occurs in two steps. The first involves the production of a lysyl-adenylate intermediate in which the amino acid is activated by its attachment to theα-phosphate of ATP. The second stage involves the transfer of the adenylate moiety to the y-phosphate of a second molecule of ATP, thereby generating Ap4A and liberating free lysine. The catalytic formation of dinucleoside polyphosphates from lysyl-adenylate intermediate is known to be promoted by Zn2+ ions although the mechanism for this process has not thus far been accounted for given the fact that the LysU amino acid sequence doesn't harbour any known Zn2+ ion consensus binding sites.In this study, we investigated the interaction between zinc ion and LysU on the structure level, intending to illuminate the potential catalytic mechanism in zinc-mediated Ap4A synthesis. The results of our investigations are reported here.First, we performed the molecular dynamics simulation with LysU comprising bound ATP substrate. Following this, binding free energies were then calculated and the potential zinc-biding sites were suggested based on computational calculations. This prediction was confirmed by protein site-directed mutagenesis with different amino acids. The mutants were over-expressed in E. coli lysU deletion strain lysU2-17A and purified using three steps as previously described. Structural characterization of mutants was used to prove integrity in advance of binding and catalysis studies with circular dichroism spectroscopy and intrinsic fluorescence titration experiments. A simple kinetic analysis of wild type LysU and mutants-mediated sequential catalysis of Ap4A was performed in the presence of Zn2+ or absence with our own built detection system. Finally, a further protein simulation was carried out to verify the binding free energy and hydrogen bond distance between zinc-binding site and ATP substrate.Our results proved that the most obvious differences between active site geometries are the relative positions of Arg269 in both sites acting as lid structure. There are other minor differences too mostly involving differential displacements of motif 2 loop amino acid residues. Interestingly, following lysyl-adenylate intermediate formation Mg2+(2) is no longer present thus leaving an anionic "pocket" near Glu264 and adenine-N7 of the lysyl-adenylate intermediate. We presumed that Glu264 could be an ideal, single amino acid residue Zn2+-binding site. Hence, we constructed total of 26 LysU single mutants including 6 Glu264 mutants,4 Arg269 mutants, and 16 inner shell/motif 2 loop mutants. Using CD spectroscopy and fluorescence titration experiments, all proteins were judged to have similar percentages of the main secondary structural elements (37%α-helix,26%β-sheet, and 38% random-coil) and unfolding transition midpoints (Cm). Therefore, in the absence of significant changes, all the LysU mutants appear to possess appropriate structural integrity relative to wild type LysU. The following zinc sensitivity assays and protein dynamics simulation support our previous presumption:the active site opening with intermediate formation, the loss of Mg2+ (2) and the opening of the anionic pocket around Glu264 forming an energy-favorable binding site for Zn2+ binding not only in simultaneous association with the a-phosphate but also adenine-N7 of lysyl-adenylate intermediate. That leaves one Zn2+ binding position free to associate with a second nucleotide substrate generating "N7-Zn(Glu264)-N7'" intermediate. Hence, theγ-phosphate of ATP attacks the enzyme-bound lysyl-adenylate, thereby forming Ap4A.Otherwise in toto, LysU has been characterized as simultaneously a lysyl-RNA synthetase (LysRS) and sequential Ap4A synthasc. However very recently, we observed LysU are found to possess new catalytic capacity, namely glycerol-kinase activity. LysU was proved to be able to catalyze glycerol-3-phosphate from glycerol and ATP in the absent of Zn2'and lysine. This kinase reaction is complementary to Zn2+-enabled Ap4A formation termed as "glycerol retard effect". Meanwhile, LysU is sensitive to hydrogen peroxide, behaving like the catalase to neutralize H2O2 although H2O2 also destroys the protein gradually too.To the best of our knowledge, LysU is the first enzyme yet characterized with at least three distinct catalytic activities and is the most diverse, multi-functional enzyme yet known. Collectively, our researches provide a solid theory foundation for the future studies of the biological functions of zinc-mediated Ap4A synthesis and LysU multi-functional research.