The catalytic mechanism of the reductive half reaction of the quinoprotein methanol dehydrogenase (MDH) is believed to proceed either through a hemiketal intermediate or by direct transfer of a hydride ion from the substrate methyl group to the cofactor, pyrroloquinoline quinone (PQQ). A crystal structure of the enzyme-substrate complex of a similar quinoprotein, glucose dehydrogenase, has recently been reported that strongly favors the hydride transfer mechanism in that enzyme. A theoretical analysis and an improved refinement of the 1.9-Å resolution crystal structure of MDH from Methylophilus methylotrophus W3A1 in the presence of methanol, reported earlier, indicates that the observed tetrahedral configuration of the C-5 atom of PQQ in that study represents the C-5-reduced form of the cofactor and lends support for a hydride transfer mechanism for MDH. Methanol and glucose dehydrogenases are two well studied quinoproteins that use 2,7,9-tricarboxypyrroloquinoline quinone (PQQ) as a cofactor ( Fig. 1A; refs. 1-4). Both enzymes require a divalent cation such as Ca 2ϩ for catalytic activity. Crystallographic studies have been reported for both methanol dehydrogenase (MDH) and glucose dehydrogenase (5-12). These crystallographic investigations have not only provided detailed information concerning the PQQ binding site, but they have also established structural frameworks for mechanistic elucidation. Two plausible mechanisms have been proposed. The first one (Fig. 1B) involves a nucleophilic addition of the alcohol to the C-5 carbonyl followed by an intramolecular retro-ene reaction (13-16). The second mechanism (Fig. 1C) involves a general base initiated hydride transfer from the alcohol to the C-5 carbonyl carbon and subsequent tautomerization of the hydride transfer intermediate to the reduced PQQH2 (15-17). Accurate structural information will be valuable in distinguishing these two proposed mechanisms.Recently, a high-resolution structure of methanol dehydrogenase from Methylophilus methylotrophus W3A1 in a second crystal form has been reported (8). Surprisingly, even when planarity constraints were applied during the structural refinement, the refined structure displayed a C-5 center significantly distorted from planarity. Although the cofactor was assigned to be the semiquinone form of PQQ, this did not provide a satisfactory explanation as to why the C-5 center is tetrahedral, because earlier quantum mechanical studies had demonstrated that the tricyclic ring in the semiquinone form of PQQ is essentially planar in the presence or absence of Ca 2ϩ (17). The C-5 methanol adduct could give a tetrahedral C-5 center, but it is not compatible with the electron density map. The tautomeric form of the reduced PQQ, in which the pyrrole H-N hydrogen is moved to the C-5 carbon, does not seem to be plausible owning to a strong intramolecular hydrogen bond interaction between the pyrrole H-N and one of the C-9 carboxylate oxygen atoms. Thus, the observed tetrahedral C-5 remained an unresolved issue. However, if one loo...
Glutathione transferases (GSTs) are a superfamily of enzymes that play a vital functional role in the cellular detoxification process. They catalyze the conjugation of the thiol group of glutathione (GSH) to the electrophilic groups of a wide range of hydrophobic substrates, leading to an easier removal of the latter from the cells. The k class is the least studied one among various classes within the superfamily. We report here the expression, purification, and crystal structure of human k class GST (hGSTK), which has been determined by the multiple-isomorphous replacement method and refined to 1.93 Å resolution. The overall structure of hGSTK is similar to the recently reported structure of k class GST from rat mitochondrion. Each subunit of the dimeric hGSTK contains a thioredoxin (TRX)-like domain and a helical domain. A molecule of glutathione sulfinate, an oxidized product of GSH, is found to bind at the G site of each monomer. One oxygen atom of the sulfino group of GSF forms a hydrogen bond with the hydroxyl group of the catalytic residue Ser16. The TRX-like domain of hGSTK shares 19% sequence identity and structure similarity with human y class GST, suggesting that the k class of GST is more closely related to the y class enzyme within the GST superfamily. The structure of the TRX-like domain of hGSTK is also similar to that of glutathione peroxidase (GPx), implying an evolutionary relationship between GST and GPx.Keywords: glutathione transferase; crystal structure; active site; glutathione sulfinate; thioredoxinlike domain; glutathione peroxidase Glutathione transferases (GSTs, EC 2.5.1.18), formerly known as glutathione S-transferases, are a superfamily of enzymes that play a vital role in cellular detoxification process. GSTs catalyze the conjugation of the thiol group of glutathione (GSH), the tripeptide g-Glu-Cys-Gly, to the electrophilic groups of a wide range of hydrophobic substrates, resulting in greater solubility and easier removal of the hydrophobic substrate from the cells (Mannervik and Danielson 1988;Pickett and Lu 1989;Coles and Ketterer 1990;Armstrong 1991;Tsuchida and Sato 1992;Wilce and Parker 1994;Sheehan et al. 2001 Abbreviations: GST, glutathione transferase; hGSTK, human k class GST; rGSTK, rat mitochondrial k class GST; GSH, glutathione; GSF, glutathione sulfinate; TRX, thioredoxin; CDNB, 1-chloro-2,4-dinitrobenzene; DTT, dithiothreitol; MIR, multiple-isomorphous replacement; NCS, noncrystallographic symmetry; RMS, root-mean-square; GPx, glutathione peroxidase; PEG, polyethylene glycol.Article published online ahead of print. Article and publication date are at
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