Structure and function of YghU, a nu-class glutathione transferase related to YfcG from Escherichia coli.
Stourman NV, Branch MC, Schaab MR, Harp JM, Ladner JE, Armstrong RN. (2011) Biochemistry 50, 1274-81. PMCID: PMC3040281
The crystal structure (1.50 Å resolution) and biochemical properties of the GSH transferase homologue, YghU, from Escherichia coli reveal that the protein is unusual in that it binds two molecules of GSH in each active site. The crystallographic observation is consistent with biphasic equilibrium binding data that indicate one tight (K(d1) = 0.07 ± 0.03 mM) and one weak (K(d2) = 1.3 ± 0.2 mM) binding site for GSH. YghU exhibits little or no GSH transferase activity with most typical electrophilic substrates but does possess a modest catalytic activity toward several organic hydroperoxides. Most notably, the enzyme also exhibits disulfide-bond reductase activity toward 2-hydroxyethyl disulfide [k(cat) = 74 ± 6 s(-1), and k(cat)/K(M)(GSH) = (6.6 ± 1.3) × 10(4) M(-1) s(-1)] that is comparable to that previously determined for YfcG. A superposition of the structures of the YghU·2GSH and YfcG·GSSG complexes reveals a remarkable structural similarity of the active sites and the 2GSH and GSSG molecules in each. We conclude that the two structures represent reduced and oxidized forms of GSH-dependent disulfide-bond oxidoreductases that are distantly related to glutaredoxin 2. The structures and properties of YghU and YfcG indicate that they are members of the same, but previously unidentified, subfamily of GSH transferase homologues, which we suggest be called the nu-class GSH transferases.
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Figure 1. Titration of the intrinsic protein fluorescence of YghU with GSH. The experimental data were fit to two binding-site model (eq. 2) with a Kd1 = 0.07 ± 0.03 mM, ΔF1 = (2.1 ± 0.6) × 105 and Kd2 = 1.3 ± 0.2 mM, ΔF2 = (-8.7 ± 0.5) × 105, r = 0.999.
Figure 2. Ribbon diagram of the structure of YghU as determined at a resolution of 1.50 Å. The two subunits are colored blue and red. The N-termini of each polypeptide are shown at the top. The C-termini of each subunit are shown at the left and right. The four molecules of GSH are illustrated as stick diagrams in each subunit.
Figure 3. Omit map of the electron density for the two molecules of GSH observed in the active site of YghU. The map, contoured at 2.5σ, was calculated with coefficients of the form Fo − Fc in which the observed and calculated structure factor amplitudes were calculated from the model lacking the coordinates of the two molecules of GSH. The final model of the two molecules is shown in stick representation. The two sulfur atoms in the middle are colored orange.
Figure 4. Details of the potential hydrogen-bonding interactions of the two GSH molecules in the active site of YghU. The primary subunit is colored blue and the opposing subunit red. The two GSH molecules are shown in stick representation. The sulfur atoms are colored yellow.
Figure 5. Overlay of the subunit structures of YfcG (blue) and YghU (green). The two GSH molecules associated with YghU and the GSSG molecule bound to YfcG are shown in stick representation. The long N-terminal extension of YghU can be seen at the top.
Figure 6. YfcG and YghU form a nu class of GSH transferases. The overall sequence similarity network contains 2851 sequences and 97144 edges. Edges represent BLAST E values of 10−18 or more stringent. Large nodes are colored by the classification of the amino acid sequence in SWISS-PROT and indicate a representative member of each subgroup as follows: alpha, GSTA3_CHICK (UniProt accession number P26697); beta, GSTB_ECOLI (P0ACA7); mu, GSTM1_RAT (P04905); nu, YFCG_ECOLI (P77526) and YGHU_ECOLI (Q46845); omega, GSTO_HUMAN (P78417); phi, GSTF1_MAIZE (P12653); pi, GSTP_ONCVO (P46427); sigma, GST_OMMSL (P46088); tau, GSTU1_ORYSJ (Q10CE7); theta, GSTT1_HUMAN (P30711); zeta, GSTZ1_HUMAN (O43708).
2011 GST Superfamily Publication
Reprinted with permission from Biochemistry.
© 2011 American Chemical Society.