Investigating the effects of posttranslational adenylylation on the metal binding sites of Escherichia coli glutamine synthetase using lanthanide luminescence spectroscopy

Abstract

Lanthanide luminescence was used to examine the effects of posttranslational adenylylation on the metal binding sites of Escherichia coli glutamine synthetase (GS). These studies revealed the presence of two lanthanide ion binding sites of GS of either adenylylation extrema. Individual emission decay lifetimes were obtained in both H 2 0 and D 2 0 solvent systems, allowing for the determination of the number of water molecules coordinated to each bound Eu3+. The results indicate that there are 4.3 2 0.5 and 4.6 ? 0.5 water molecules coordinated to Eu3+ bound to the nl site of unadenylylated enzyme, GSo, and fully adenylylated enzyme, GS12, respectively, and that there are 2.6 ? 0.5 water molecules coordinated to Eu3+ at site n2 for both GSo and GSl2. Energy transfer measurements between the lanthanide donor-acceptor pair Eu3+ and Nd3+, obtained an intermetal distance measurement of 12.1 ? 1.5 A. Distances between a Tb" ion at site n2 and tryptophan residues were also performed with the use of single-tryptophan mutant forms of E. coli GS. The dissociation constant for lanthanide ion binding to site nl was observed to decrease from Kd = 0.35 2 0.09 pM for GSo to Kd = 0.06 ? 0.02 pM for GSI2. The dissociation constant for lanthanide ion binding to site n2 remained unchanged as a function of adenylylation state; Kd = 3.8 2 0.9 pM and Kd = 2.6 2 0.7 pM for GSo and GSI2, respectively. Competition experiments indicate that Mn2+ affinity at site nl decreases as a function of increasing adenylylation state, from Kd = 0.05 2 0.02 pM for GSo to K d = 0.35 ? 0.09 pM for GSI2. Mn2+ affinity at site n2 remains unchanged (Kd = 5.3 ? 1.3 pM for GSo and Kd = 4.0 ? 1.0 pM for GSI2). The observed divalent metal ion affinities, which are affected by the adenylylation state, agrees with other steady-state substrate experiments (Abell LM, Villafranca JJ, 1991. Biochemistry 30: 1413-141 8), supporting the hypothesis that adenylylation regulates GS by altering substrate and metal ion affinities.

Discovery of a New Class of Immunosuppressants from Trichothecium roseum Co‐inspired by Cross‐Kingdom Similarity in Innate Immunity and Pharmacophore Motif

“…The Eu III emissions in 3 and 4 are enhanced in CD 2 Cl 2 compared with that in CH 2 Cl 2 . However, the Eu III emissions are quenched to some extent in the mixed solvents containing CH 2 Cl 2 and methanol (1:1 v/v) compared with that in neat dichloromethane 15a–15c. It is noted that the intensity of the 5 D 0 → 7 F 2 transition is enhanced by almost five times in 3 compared with that in 4 in all the solvents (Table 2).…”

Adenylylation and Catalytic Properties of Mycobacterium tuberculosis Glutamine Synthetase Expressed in Escherichia coli versus Mycobacteria

“…1). The validity of this strategy was established first with E. coli GS, wherein the previously described strains YMC21E and YMC21D produce fully unadenylylated and fully adenylylated GS, respectively (25,34). Mass spectra for E. coli GS in the fully adenylylated, fully unadenylylated, and SVPDE-treated states are shown in Fig.…”

“…The YMC21E also lacks ATase, whereas YMC21D lacks the uridyl transferase that modulates the adenylylation/ deadenylylation rates of ATase. Culture conditions for the expression of E. coli GS or MTb GS in these strains has been described previously (10,25).…”

“…effect of nitrogen levels on the adenylylation process. Specifically, cells were grown in standard media as described under "Materials and Methods" (25), or the media were supplemented with 20 mM glutamine. For E. coli (other than YMC21E), the addition of glutamine to the culture media yields highly adenylylated GS (19,23,35).…”

“…In many bacteria, the adenylylation process involves a highly regulated enzymatic cascade that responds to environmental nitrogen levels (20 -22). For Escherichia coli GS, the biochemical effects of adenylylation on GS catalytic activity are well characterized (20 -22) and include changes in the affinity of the enzyme for metal ion cofactors and substrates (24,25). Disruption of the glnE gene in MTb yields a lethal phenotype (26,27), suggesting that GS adenylylation is a critical process in Mycobacteria also.…”