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An x-ray study indicated that microsomal prostaglandin E synthase type 2 (mPGES2) is a heme-bound protein and catalyzes prostaglandin (PG) H2 degradation, but not PGE2 formation (Yamada, T., and Takusagawa, F. (2007) Biochemistry 46, 8414-8424). In response to the x-ray study, Watanabe et al. claimed that mPGES2 is a heme-free protein and that both the heme-free and heme-bound proteins have PGE2 synthesis activity in the presence of dithiothreitol (Watanabe, K., Ito, S., and Yamamoto, S. (2008) Biochem. Biophys. Res. Commun. 367, 782-786). To resolve the contradictory results, the heme-binding scheme of mPGES2 was further characterized in vivo and in vitro by absorption and fluorescence spectroscopies. A substantial amount of heme-bound mPGES2 was detected in cell extracts. The heme content in mPGES2 was increased along with an increase in Fe(3+) in the culture medium. Heme-free mPGES2 was converted to the heme-bound form by mixing it with pig liver extract, indicating that mPGES2 is capable of forming a complex with heme in mammalian cells. Heme binds to mPGES2 only in the presence of glutathione. The newly determined heme dissociation constant (2.9 nM) supports strongly that mPGES2 is a heme-bound protein in vivo. The bound heme was not dissociated by oxidation by H2O2 or reduction by glutathione or 2-mercaptoethanol. However, reduction by dithiothreitol (an artificial reducing compound) induced the bound heme to dissociate from mPGES2 and released heme-free mPGES2, which exhibited PGE2 synthesis activity in vitro. Imidazole bound to mPGES2 by stacking on the bound heme and inhibited heme oxidation by H2O2 and reduction by dithiothreitol.

Objective: To investigate the diuretic effects, tolerability, and adverse effects of furosemide and torsemide after short- and long-term administration in healthy dogs., Animals: 8 mixed-breed dogs., Procedures: In a crossover study, furosemide (2 mg/kg), torsemide (0.2 mg/kg), or placebo (bifidobacterium [1 mg/kg]) was administered orally to each dog every 12 hours for 14 days. Blood and urine samples were collected before the study (baseline data) and at intervals on the 1st (short-term administration) and 14th day (long-term administration) of treatment for assessment of urine volume and specific gravity and selected clinicopathologic variables including BUN, creatinine, and aldosterone concentrations, and creatinine clearance., Results: Compared with the baseline value, short-term administration of furosemide or torsemide immediately increased urine volume significantly; after long-term administration of either drug, urine specific gravity decreased significantly. Compared with the effect of placebo, the 24-hour urine volume was significantly increased after short-term administra-tion of furosemide or torsemide. In addition, it was significantly increased after long-term administration of torsemide, compared with that of short-term administration. Long-term administration of furosemide or torsemide increased the BUN and plasma creatinine con-centrations, compared with the baseline value. Compared with the baseline value, plasma aldosterone concentration was significantly increased after long-term administration of either drug and was significantly higher after torsemide treatment than after furosemide treatment., Conclusions and Clinical Relevance: In dogs, diuretic resistance developed after 14 days of furosemide, but not torsemide, administration; however, both loop diuretics were associated with increased BUN and plasma creatinine concentrations, compared with values before treatment.

Cytolethal distending toxin (CDT) secreted by Actinobacillus actinomycetemcomitans induces cell cycle arrest of cultured cells in the G2 phase. The crystal structure of the natural form of A. actinomycetemcomitans DCT (aCDT) has been determined at 2.4 A resolution. aCDT is a heterotrimer consisting of the three subunits, aCdtA, aCdtB, and aCdtC. Two crystallographically independent aCDTs form a dimer through interactions of the aCdtB subunits. The primary structure of aCDT has 94.3% identity with that of Haemophilus ducreyi CDT (hCDT), and the structure of aCDT is quite similar to that of hCDT reconstituted from the three subunits determined recently. However, the molecular packings in the crystal structures of aCDT and hCDT are quite different. A careful analysis of molecular packing suggests that variation of the amino acid residues in a nonconserved loop (181TSSPSSPERRGY192 of aCdtB and 181NSSSSPPERRVY192 of hCdtB) is responsible for the different oligomerization of very similar CDTs. The loop of aCdtB has a conformation to form a dimer, while the loop conformation of hCdtB prevents hCDT from forming a dimer. Although dimerization of aCDT does not affect toxic activity, it changes the stability of protein. aCDT rapidly aggregates and loses toxic activity in the absence of sucrose in a buffered solution, while hCDT is stable and retains toxic activity. Another analysis of crystal structures of aCDT and hCDT suggests that the receptor contact area is in the deep groove between CdtA and CdtC, and the characteristic "aromatic patch" on CdtA.

S-Adenosylhomocysteine hydrolase (AdoHcyase) catalyzes the hydrolysis of S-adenosylhomocysteine to form adenosine and homocysteine. On the bases of crystal structures of the wild type enzyme and the D244E mutated enzyme complexed with 3'-keto-adenosine (D244E.Ado*), we have identified the important amino acid residues, Asp-130, Lys-185, Asp-189, and Asn-190, for the catalytic reaction and have proposed a catalytic mechanism (Komoto, J., Huang, Y., Gomi, T., Ogawa, H., Takata, Y., Fujioka, M., and Takusagawa, F. (2000) J. Biol. Chem. 275, 32147-32156). To confirm the proposed catalytic mechanism, we have made the D130N, K185N, D189N, and N190S mutated enzymes and measured the catalytic activities. The catalytic rates (k(cat)) of D130N, K185N, D189N, and N190S mutated enzymes are reduced to 0.7%, 0.5%, 0.1%, and 0.5%, respectively, in comparison with the wild type enzyme, indicating that Asp-130, Lys-185, Asp-189, and Asn-190 are involved in the catalytic reaction. K(m) values of the mutated enzymes are increased significantly, except for the N190S mutation, suggesting that Asp-130, Lys-185, and Asp-189 participate in the substrate binding. To interpret the kinetic data, the oxidation states of the bound NAD molecules of the wild type and mutated enzymes were measured during the catalytic reaction by monitoring the absorbance at 340 nm. The crystal structures of the WT and D244E.Ado*, containing four subunits in the crystallographic asymmetric unit, were re-refined to have the same subunit structures. A detailed catalytic mechanism of AdoHcyase has been revealed based on the oxidation states of the bound NAD and the re-refined crystal structures of WT and D244E.Ado*. Lys-185 and Asp-130 abstract hydrogen atoms from 3'-OH and 4'-CH, respectively. Asp-189 removes a proton from Lys-185 and produces the neutral N zeta (-NH(2)), and Asn-190 facilitates formation of the neutral Lys-185. His-54 and His-300 hold and polarize a water molecule, which nucleophilically attacks the C5'- of 3'-keto-4',5'-dehydroadenosine to produce 3'-keto-Ado.

D-eritadenine (DEA) is a potent inhibitor (IC(50) = 7 nm) of S-adenosyl-l-homocysteine hydrolase (AdoHcyase). Unlike cyclic sugar Ado analogue inhibitors, including mechanism-based inhibitors, DEA is an acyclic sugar Ado analogue, and the C2' and C3' have opposite chirality to those of the cyclic sugar Ado inhibitors. Crystal structures of DEA alone and in complex with AdoHcyase have been determined to elucidate the DEA binding scheme to AdoHcyase. The DEA-complexed structure has been analyzed by comparing it with two structures of AdoHcyase complexed with cyclic sugar Ado analogues. The DEA-complexed structure has a closed conformation, and the DEA is located near the bound NAD(+). However, a UV absorption measurement shows that DEA is not oxidized by the bound NAD(+), indicating that the open-closed conformational change of AdoHcyase is due to the substrate/inhibitor binding, not the oxidation state of the bound NAD. The adenine ring of DEA is recognized by four essential hydrogen bonds as observed in the cyclic sugar Ado complexes. The hydrogen bond network around the acyclic sugar moiety indicates that DEA is more tightly connected to the protein than the cyclic sugar Ado analogues. The C3'-H of DEA is pointed toward C4 of the bound NAD(+) (C3'...C4 = 3.7 A), suggesting some interaction between DEA and NAD(+). By placing DEA into the active site of the open structure, the major forces to stabilize the closed conformation of AdoHcyase are identified as the hydrogen bonds between the backbone of His-352 and the adenine ring, and the C3'-H...C4 interaction. DEA has been believed to be an inactivator of AdoHcyase, but this study indicates that DEA is a reversible inhibitor. On the basis of the complexed structure, selective inhibitors of AdoHcyase have been designed.

A site-directed mutagenesis, D244E, of S-adenosylhomocysteine hydrolase (AdoHcyase) changes drastically the nature of the protein, especially the NAD(+) binding affinity. The mutant enzyme contained NADH rather than NAD(+) (Gomi, T., Takata, Y., Date, T., Fujioka, M., Aksamit, R. R., Backlund, P. S., and Cantoni, G. L. (1990) J. Biol. Chem. 265, 16102-16107). In contrast to the site-directed mutagenesis study, the crystal structures of human and rat AdoHcyase recently determined have shown that the carboxyl group of Asp-244 points in a direction opposite to the bound NAD molecule and does not participate in any hydrogen bonds with the NAD molecule. To explain the discrepancy between the mutagenesis study and the x-ray studies, we have determined the crystal structure of the recombinant rat-liver D244E mutant enzyme to 2.8-A resolution. The D244E mutation changes the enzyme structure from the open to the closed conformation by means of a approximately 17 degrees rotation of the individual catalytic domains around the molecular hinge sections. The D244E mutation shifts the catalytic reaction from a reversible to an irreversible fashion. The large affinity difference between NAD(+) and NADH is mainly due to the enzyme conformation, but not to the binding-site geometry; an NAD(+) in the open conformation is readily released from the enzyme, whereas an NADH in the closed conformation is trapped and cannot leave the enzyme. A catalytic mechanism of AdoHcyase has been proposed on the basis of the crystal structures of the wild-type and D244E enzymes.

A pCW vector harboring rat liver serine dehydratase cDNA was expressed in Escherichia coli. The expressed level was about 5-fold higher in E. coli BL21 than in JM109 cell extract; the former lacked two kinds of proteases. Immunoblot analysis revealed the occurrence of a derivative other than serine dehydratase in the JM109 cell extract. The recombinant enzyme was purified to homogeneity. Staphylococcus aureus V8 protease and trypsin cleaved the enzyme at Glu-206 and Lys-220, respectively, with a concomitant loss of enzyme activity. Spectrophotometrically, the nicked enzyme showed a approximately 50% reduced capacity for binding of the coenzyme pyridoxal phosphate and no spectral change of circular dichroism in the region at 300-480 nm, whereas circular dichroism spectra of both enzymes in the far-UV region were similar, suggesting that proteolysis impairs the coenzyme binding without an accompanying gross change of the secondary structure. Whereas the nicked enzyme behaved like the intact enzyme on Sephadex G-75 column chromatography, it was dissociated into two fragments on the column containing 6 M urea. Upon the removal of urea, both fragments spontaneously refolded. These results suggest that serine dehydratase consists of two folding domains connected by a region that is very susceptible to proteases.

The structure of S-adenosylmethionine synthetase (MAT, ATP:L-methionine S-adenosyltransferase, EC 2.5.1.6.) from Escherichia coli has been determined at 3.0 A resolution by multiple isomorphous replacement using a uranium derivative and the selenomethionine form of the enzyme (SeMAT). The SeMAT data (9 selenomethionine residues out of 383 amino acid residues) have been found to have a sufficient phasing power to determine the structure of the 42,000 molecular weight protein by combining them with the other heavy atom derivative data (multiple isomorphous replacement). The enzyme consists of four identical subunits; two subunits form a spherical tight dimer, and pairs of these dimers form a peanut-shaped tetrameric enzyme. Each pair dimer has two active sites which are located between the subunits. Each subunit consists of three domains that are related to each other by pseudo-3-fold symmetry. The essential divalent (Mg2+/Co2+) and monovalent (K+) metal ions and one of the product, Pi ions, were found in the active site from three separate structures.

5,6,7,7a,9,10,14b,14c-Octahydro-4-[2,3-O-(1-methylethylidene )-beta-D- ribofuranosyl]cyclopenta-[4,5]pyrimido[5',4':3,4]pyrrolo[2,1- a]isoquinoline-1,3(2H,4H)-dione, C25H31N3O6, M(r) = 469.54, tetragonal, P4(3)2(1)2, a = 12.577 (2), b = 12.577 (2), c = 29.893 (4) A, V = 4729 (1) A3, Z = 8, Dx = 1.319 g cm-3, lambda (Cu K alpha) = 1.5418 A, mu = 7.9 cm-1, F(000) = 2000, T = 298 K, final R = 0.038, wR = 0.064 for all 1884 independent reflections and 432 variables. The crystal structure shows a syn conformation around the N(1)--C(1') single bond, consistent with other C(6)-substituted uridine analogs, and an unusual O(1') endo conformation of the ribose ring. The stereochemistries of the three newly created chiral centers are 14cR, 4aR, 7aS.

(+/-)-exo-1,4-Epoxy-2-formamido-1,2,3,4-tetrahydro-8- trifluoromethylnaphthalene (1), C12H10F3NO2, Mr = 257.21, Pccn, a = 8.895 (1), b = 19.968 (5), c = 12.656 (3) A, V = 2247.9 (8) A3, Z = 8, Dx = 1.520 g cm-3, lambda(Mo K alpha) = 0.71069 A, mu = 1.49 cm-1, F(000) = 1056, T = 297 K, R = 0.0466 for 1481 independent reflections collected. The torsion angle for N(10)-C(2)-C(1)-C(8a) is 167.4 (2) degrees. (+/-)-exo-1,4-Epoxy-2-formamido-1,2,3,4-tetrahydro-6- trifluoromethylnaphthalene, (2), C12H10F3NO2, Mr = 257.21, P2(1)2(1)2(1), a = 8.52 (2), b = 26.15 (2), c = 5.06 (5) A, V = 1127 (11) A3, Z = 4, Dx = 1.516 g cm-3, lambda(Mo K alpha) = 0.71069 A, mu = 1.29 cm-1, F(000) = 528, T = 297 K, R = 0.108 for 932 independent reflections collected. The torsion angle N(10)-C(2)-C(1)-C(8a) is 172.5 (6) degrees; the formamido group in both (1) and (2) is exo. X-ray studies on (1) suggest a hydrogen bond between N(10) and O(12) and similarly for (2).

[Ni(C13H14N4)], Mr = 284.98, monoclinic, P2(1)/c, a = 10.096 (2), b = 9.360 (2), c = 30.565 (4) A, beta = 119.45 (2) degrees, V = 2515.1 (9) A3, Z = 8, Dx = 1.505 g cm-3, Cu K alpha, lambda = 1.5418 A, mu = 20.22 cm-1, F(000) = 1168, T = 298 (2) K, 437 parameters refined, final R = 0.038 for all 2587 reflections. The complex takes a square-planar geometry around the Ni atom. Although the coordination geometries of the two crystallographically independent complexes are quite similar to each other, significant differences in planarity and Ni-N distances are observed.

Crystals of a 2:1 complex between the self-complementary DNA hexamer d(ATGCAT) and the antitumor drug actinomycin D have been grown from solutions of polyethylene glycol 400. The crystals are orthorhombic with space group P2(1)2(1)2(1) and a = 95.6, b = 42.7, and c = 40.8 A. A Patterson map calculated from preliminary diffractometer data as well as packing considerations suggest a model in which the actinomycin D is intercalated into a double-stranded DNA hexamer. There are four such complexes in the asymmetric unit.