Comparison of the functions of glutathionylspermidine synthetase/amidase from E. coli and its predicted homologues YgiC and YjfC

Abstract

Protein function prediction is very important in establishing the roles of various proteins in bacteria; however, some proteins in the E. coli genome have their function assigned based on low percent sequence homology that does not provide reliable assignments. We have made an attempt to verify the prediction that E. coli genes ygiC and yjfC encode proteins with the same function as glutathionylspermidine synthetase/amidase (GspSA). GspSA is a bifunctional enzyme that catalyzes the ATP-dependent formation and hydrolysis of glutathionylspermidine (G-Sp), a conjugate of glutathione (GSH) and spermidine. YgiC and YjfC proteins show 51% identity between themselves and 28% identity to the synthetase domain of the GspSA enzyme. YgiC and YjfC proteins were expressed and purified, and the properties of GspSA, YgiC, and YjfC were compared. In contrast to GspSA, proteins YgiC and YjfC did not bind to G-Sp immobilized on the affinity matrix. We demonstrated that all three proteins (GspSA, YgiC and YjfC) catalyze the hydrolysis of ATP; however, YgiC and YjfC cannot synthesize G-Sp, GSH, or GSH intermediates. gsp, ygiC, and yjfC genes were eliminated from the E. coli genome to test the ability of mutant strains to synthesize G-Sp conjugate. E. coli cells deficient in GspSA do not produce G-Sp while synthesis of the conjugate is not affected in ΔygiC and ΔyjfC mutants. All together our results indicate that YgiC and YjfC are not glutathionylspermidine synthetases as predicted from the amino acid sequence analysis.

Keywords: ATP-grasp domain; ATPase; Glutathione; glutathionylspermidine; glutathionylspermidine synthetase/amidase.

Figure 1

Sequence alignment of GspSA synthetase domain, YgiC, and YjfC. The star (*) indicates fully conserved residue, colon (:) indicates conservation between groups of strongly similar properties, single dot (•) indicates conservation between groups of weakly similar properties. The shaded amino acid residues were found to participate in the binding of ATP by GspSA [7]. The amino acid residues in boxes represent conserved residues of ATP-grasp fold, R316, K498, K533. The amino acid residues highlighted in yellow are essential residues for substrates binding by GspSA as determined in [7].

Figure 2

ATPase activity assay. The ATPase activities of GspSA, YgiC, and YjfC were determined by enzyme PK/LDH coupled assay. The change of absorbance at 340 nm due to oxidation of NADH was used to calculate the rate of hydrolysis of ATP. The initial concentration of ATP was 10 mM, the concentrations of YgiC and YjfC were varied between 0.01 and 5 μM, GspSA was varied between 0.2 and 16 μM.

Figure 3

Ligase activity assay of GspSA, YgiC, and YjfC. The substrate composition for the reactions was: A. cysteine and glutamate, B. GSH and spermidine, C. γ-glutamylcysteine and spermidine, D. γ-glutamylcysteine and glycine. The reactions were stopped 3 hours after the addition of ATP by brief heating, derivatized with DTNB, and the thiol content of the reaction mixtures was analyzed by reverse-phase HPLC. Labeled peaks represent DTNB derivatives of corresponding thiols. Peaks at 18 min and 21 min are 2-nitro-5-thiobenzoate and the excess of DTNB correspondently.

Figure 4

SDS-PAGE analysis of fractions collected from G-Sp affinity column. Lanes: 1 – fraction containing proteins that did not bind to the column; 2 – initial wash with 50 mM potassium phosphate buffer, pH 7.5; 3 – molecular marker; 4 – 0.2 M NaCl, 50 mM potassium phosphate buffer, pH 7.5; 5 – 0.6 M NaCl, 50 mM potassium phosphate buffer, pH 7.5; 6 - 1 M NaCl, 50 mM potassium phosphate buffer, pH 7.5; 7, 8 - 1% 2-mercaptoethanol, 50 mM potassium phosphate buffer, pH 7.5.

Figure 5

Analysis of thiol content in E. coli cells. Analysis of the thiol content of wild type and gsp, yjfC, and ygiC gene knockout E. coli strains grown in LB media to stationary phase under anaerobic conditions was performed using HPLC. Labeled peaks represent DTNB derivatives of G-Sp and GSH. Peak immediately followed GSH (14 min) corresponds to DTNB derivative of γ-glutamylcysteine. Peaks at 18 min and 21 min are 2-nitro-5-thiobenzoate and the excess of DTNB correspondently.