Three-dimensional structure, catalytic properties, and evolution of a sigma class glutathione transferase from squid, a progenitor of the lens S-crystallins of cephalopods

Abstract

The glutathione transferase from squid digestive gland is unique in its very high catalytic activity toward 1-chloro-2,4-dinitrobenzene and in its ancestral relationship to the genes encoding the S-crystallins of the lens of cephalopod eye. The three-dimensional structure of this glutathione transferase in complex with the product 1-(S-glutathionyl)-2,4-dinitrobenzene (GSDNB) has been solved by multiple isomorphous replacement techniques at a resolution of 2.4 A. Like the cytosolic enzymes from vertebrates, the squid protein is a dimer. The structure is similar in overall topology to the vertebrate enzymes but has a dimer interface that is unique when compared to all of the vertebrate and invertebrate structures thus far reported. The active site of the enzyme is very open, a fact that appears to correlate with the high turnover number (800 s-1 at pH 6.5) toward 1-chloro-2,4-dinitrobenzene. Both kcat and kcat/KmCDNB exhibit pH dependencies consistent with a pKa for the thiol of enzyme-bound GSH of 6.3. The enzyme is not very efficient at catalyzing the addition of GSH to enones and epoxides. This particular characteristic appears to be due to the lack of an electrophilic residue at position 106, which is often found in other GSH transferases. The F106Y mutant enzyme is much improved in catalyzing these reactions. Comparisons of the primary structure, gene structure, and three-dimensional structure with class alpha, mu, and pi enzymes support placing the squid protein in a separate enzyme class, sigma. The unique dimer interface suggests that the class sigma enzyme diverged from the ancestral precursor prior to the divergence of the precursor gene for the alpha, mu, and pi classes.

The crystal structure of the nitrogen regulation fragment of the yeast prion protein Ure2p

“…Both the wild-type pi class GST and an enzymatically inactivate mutant were shown to inhibit activation of the nuclear transcriptional activating protein jun by jun kinase (JNK), with the GST's C-terminal residues (194 to 201) being implicated as important for this inhibitory activity. The S-crystallins of cephalopods, which perform a refractive function in the eye lens, have evolved from a sigma class GST (48,49).…”

Tyrosine 8 contributes to catalysis but is not required for activity of rat liver glutathione S‐transferase, 1–1

“…structures of representatives of the a class (Sinning et al, 1993), p class Raghunathan et al, 1994), T class (Reinemer et al, 1991(Reinemer et al, , 1992Garcia-Saez et al, 1994), and u class (Ji et al, 1995) have been determined from X-ray crystallography. The tyrosine residue near the N-terminal end of the enzyme is conserved in all known mammalian cytosolic glutathione S-transferases and is considered to be essential for catalysis (Wilce & Parker, 1994).…”

Glutathione S-Transferase Pi Has at Least Three Distinguishable Xenobiotic Substrate Sites Close to Its Glutathione-binding Site

“…The catalytic diversity of mammalian cytosolic GSTs, which can exist as homo-or heterodimers, arises in part from the existence of at least eight distinct classes (named Alpha, Kappa, Mu, Omega, Pi, Sigma, Theta, and Zeta) (1,4). The glutathione-binding site (G-site) and the catalytic mechanism of these enzymes have been the targets of many investigations involving chemical modification (5)(6)(7)(8), site-directed mutagenesis (9 -14), and x-ray crystallographic analysis (15)(16)(17)(18)(19)(20)(21). On the contrary, the electrophilic substrate-binding site (H-site) of GSTs is more complex and is class-specific.…”

“…On the contrary, the electrophilic substrate-binding site (H-site) of GSTs is more complex and is class-specific. Crystallographic studies have shown that the H-site is quite dissimilar among different classes of GSTs (15)(16)(17)(18)(19)(20)(21). For example, in GST M1-1, the H-site is a hydrophobic cavity, whereas in GST P1-1 (15), the H-site is half-hydrophobic and half-hydrophilic with functionally important water molecules (16).…”