Biomedical subjects

F Takusagawa

Publications and source records attributed to F Takusagawa.

At least 19 recordsLinked to original sources

PubMed2001

Anti-leukemia selectivity in actinomycin analogues.

An excellent anti-leukemia activity has been found in a group of actinomycin D analogues derivatized at the 2,2'- or 5,5'-position of the depsipeptides. On the basis of the water solubilities, the DNA binding affinities, the RNA synthesis inhibitory activities, the anticancer activities of actinomycin D (AMD), and the crystal structures of DNA-AMD complexes, it becomes clear that AMD is extremely well designed as an effective poison produced by micro-organisms. The anticancer activity of AMD is mainly due to its selective inhibition of RNA synthesis. We have hypothesized that a modification on the AMD structure at a site not involved in DNA interaction can either increase or decrease the diffusion rate of the analogue into certain cancer cells. Since the i-propyl groups of the D-valine residues at the 2,2'-positions and N-methyl-L-valine residues at the 5,5'-positions in the depsipeptides do not participate in interaction with DNA, these amino acid residues were replaced with other D-amino acid residues and N-methyl-L-amino acid residues, respectively. The cancer screen tests have indicated that AMD analogues 2,2'-D-PheAMD, 2,2'-D-OmeAMD, 5,5'-L-TyrAMD, 5,5'-D-ValAMD, 5,5'-D-TyrAMD, 5,5'-D-PheAMD, and 5,5'-D-OmeAMD, inhibit selectively the growth of leukemia cell lines at about 100- to 500-fold lower drug concentrations than those required to inhibit other cancer cell lines.

Animals↗

PubMed2000

Effects of site-directed mutagenesis on structure and function of recombinant rat liver S-adenosylhomocysteine hydrolase. Crystal structure of D244E mutant enzyme.

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.

Adenosylhomocysteinase↗

PubMed2000

Natural and synthetic analogues of actinomycin D as Grb2-SH2 domain blockers.

Natural analogues (D, C2, and VII) of actinomycin inhibit Grb2 SH2 domain binding with phosphopeptide-derived from Shc in vitro and in intracellular system. To study structure-activity relationships, 13 actinomycin analogues were synthesized and we found that the inhibition activity depended on the substituents of cyclic peptide groups in actinomycin and two analogues with Tyr residue are the most potent inhibitors with IC50 value of 0.5 and 0.8 microM, respectively.

Adaptor Proteins, Signal Transducing↗

PubMed2000

Mechanisms for auto-inhibition and forced product release in glycine N-methyltransferase: crystal structures of wild-type, mutant R175K and S-adenosylhomocysteine-bound R175K enzymes.

Glycine N-methyltransferase (S-adenosyl-l-methionine: glycine methyltransferase, EC 2.1.1.20; GNMT) catalyzes the AdoMet-dependent methylation of glycine to form sarcosine (N-methylglycine). Unlike most methyltransferases, GNMT is a tetrameric protein showing a positive cooperativity in AdoMet binding and weak inhibition by S-adenosylhomocysteine (AdoHcy). The first crystal structure of GNMT complexed with AdoMet showed a unique "closed" molecular basket structure, in which the N-terminal section penetrates and corks the entrance of the adjacent subunit. Thus, the apparent entrance or exit of the active site is not recognizable in the subunit structure, suggesting that the enzyme must possess a second, enzymatically active, "open" structural conformation. A new crystalline form of the R175K enzyme has been grown in the presence of an excess of AdoHcy, and its crystal structure has been determined at 3.0 A resolution. In this structure, the N-terminal domain (40 amino acid residues) of each subunit has moved out of the active site of the adjacent subunit, and the entrances of the active sites are now opened widely. An AdoHcy molecule has entered the site occupied in the "closed" structure by Glu15 and Gly16 of the N-terminal domain of the adjacent subunit. An AdoHcy binds to the consensus AdoMet binding site observed in the other methyltransferase. This AdoHcy binding site supports the glycine binding site (Arg175) deduced from a chemical modification study and site-directed mutagenesis (R175K). The crystal structures of WT and R175K enzymes were also determined at 2.5 A resolution. These enzyme structures have a closed molecular basket structure and are isomorphous to the previously determined AdoMet-GNMT structure. By comparing the open structure to the closed structure, mechanisms for auto-inhibition and for the forced release of the product AdoHcy have been revealed in the GNMT structure. The N-terminal section of the adjacent subunit occupies the AdoMet binding site and thus inhibits the methyltransfer reaction, whereas the same N-terminal section forces the departure of the potentially potent inhibitor AdoHcy from the active site and thus facilitates the methyltransfer reaction. Consequently GNMT is less active at a low level of AdoMet concentration, and is only weakly inhibited by AdoHcy. These properties of GNMT are particularly suited for regulation of the cellular AdoMet/AdoHcy ratio.

Allosteric Regulation↗

PubMed2000

Crystallization and preliminary X-ray diffraction studies of d(ACGTAGCTACGT)2:[actinomycin D, (echinomycin)2] and d(ACGTAGCTACGT)2:[actinomycin D, (triostin A)2] complexes.

A DNA-multiple drug complex, d(ACGTAGCTACGT)(2):[actinomycin D, (echinomycin)(2)] has been crystallized. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 85.6, b = 72.8, c = 56.6 A, beta = 101.5 degrees at 93 K and Z = 8. The crystal diffracted to 3.0 A resolution along the DNA fiber axis and to 3.5 A resolution in other directions. The Patterson maps indicate that all complexes in the crystal are oriented along their helical axes in the [101;] direction.

Crystallization↗

PubMed1999

Crystal structure of S-adenosylhomocysteine hydrolase from rat liver.

The crystal structure of rat liver S-adenosyl-L-homocysteine hydrolase (AdoHcyase, EC 3.3.1.1) which catalyzes the reversible hydrolysis of S-adenosylhomocysteine (AdoHcy) has been determined at 2.8 A resolution. AdoHcyase from rat liver is a tetrameric enzyme with 431 amino acid residues in each identical subunit. The subunit is composed of the catalytic domain, the NAD+-binding domain, and the small C-terminal domain. Both catalytic and NAD+-binding domains are folded into an ellipsoid with a typical alpha/beta twisted open sheet structure. The C-terminal section is far from the main body of the subunit and extends into the opposite subunit. An NAD+ molecule binds to the consensus NAD+-binding cleft of the NAD+-binding domain. The peptide folding pattern of the catalytic domain is quite similar to the patterns observed in many methyltransferases. Although the crystal structure does not contain AdoHcy or its analogue, there is a well-formed AdoHcy-binding crevice in the catalytic domain. Without introducing any major structural changes, an AdoHcy molecule can be placed in the catalytic domain. In the structure described here, the catalytic and NAD+-binding domains are quite far apart from each other. Thus, the enzyme appears to have an "open" conformation in the absence of substrate. It is likely that binding of AdoHcy induces a large conformational change so as to place the ribose moiety of AdoHcy in close proximity to the nicotinamide moiety of NAD+. A catalytic mechanism of AdoHcyase has been proposed on the basis of this crystal structure. Glu155 acts as a proton acceptor from the O3'-H when the proton of C3'-H is abstracted by NAD+. His54 or Asp130 acts as a general acid-base catalyst, while Cys194 modulates the oxidation state of the bound NAD+. The polypeptide folding pattern of the catalytic domain suggests that AdoHcy molecules can travel freely to and from AdoHcyase and methyltransferases to properly regulate methyltransferase activities. We believe that the crystal structure described here can provide insight into the molecular architecture of this important regulatory enzyme.

Adenosylhomocysteinase↗

PubMed1999

Rat liver serine dehydratase. Bacterial expression and two folding domains as revealed by limited proteolysis.

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.

Animals↗

PubMed1999

Crystallization and preliminary x-ray diffraction studies of guanidinoacetate methyltransferase from rat liver.

Guanidinoacetate methyltransferase is the enzyme which catalyzes the last step of creatine biosynthesis. The enzyme is found ubiquitously and in abundance in the livers of all vertebrates. Recombinant rat-liver guanidinoacetate methyltransferase has been crystallized with guanidinoacetate and S-adenosylhomocysteine. The crystals belong to the monoclinic space group P2(1), with unit-cell parameters a = 54.8, b = 162.5, c = 56.1 A, beta = 96.8 (1) degrees at 93 K, and typically diffract beyond 2.8 A.

Animals↗

PubMed1998

Structure, function and physiological role of glycine N-methyltransferase.

Glycine N-methyltransferase (EC 2.1.1.20) catalyzes the transfer of the methyl group of S-adenosylmethionine (AdoMet) to glycine to form S-adenosylhomocysteine and sarcosine. Unlike most AdoMet-dependent methyltransferases, glycine N-methyltransferase is a tetramer of identical subunits. Crystallography of recombinant rat glycine N-methyltransferase indicates that four nearly spherical subunits are arranged to form a flat, square tetramer with a large hole in the centre. The enzyme occurs abundantly in the livers of rat, rabbit and mouse. Glycine N-methyltransferases from rat, rabbit, human and pig livers are shown to have similar amino acid sequences and, with the enzymes from rat and rabbit livers, it is demonstrated that the N-terminal valine is acetylated. Glycine N-methyltransferases from livers exhibit sigmoidal rate behaviour with respect to AdoMet and hyperbolic behaviour with respect to glycine at all pH tested. However, recombinant rat glycine N-methyltransferase which lacks the N-terminal acetyl group shows no cooperativity toward AdoMet at neutral pH, suggesting that elimination of the positive charge at the N-terminus is required for cooperative behaviour. Glycine N-methyltransferase binds 5-methyltetrahydropteroylpentaglutamate tightly, resulting in inhibition of the catalytic activity. The nature of these unique functional features is discussed in the light of the three-dimensional structure of the enzyme. The tissue and subcellular localization of the enzyme and its possible role in methionine metabolism are reviewed.

Amino Acid Sequence↗

PubMed1997

Selectivity of F8-actinomycin D for RNA:DNA hybrids and its anti-leukemia activity.

Although many compounds have been found that bind to DNA in various ways and exhibit various biological activities, few compounds that specifically bind to RNA or RNA:DNA hybrids are known, even though such compounds are expected to have important biological properties. For example, one characteristic function of the retroviruses, which is generally not found in eukaryotic cells, is the production of an RNA:DNA hybrid in the viral replication phase. If an agent is designed to bind only to an RNA:DNA hybrid, and not to DNA or to RNA, such an agent might be able to inhibit specifically the RNase H activity of retroviral reverse transcriptase, and therefore suppress viral replication. Actinomycin D is known to bind to double-stranded DNA, but not to RNA, because steric hindrance between the 2-amino group of the phenoxazone ring and the 2'-hydroxyl group of RNA prevents intercalation of the compound. However, if the > C-H moiety at the 8-position of the phenoxazone ring is replaced by a > C-F, a possible hydrogen-bond acceptor, this analogue (8-fluoro-actinomycin D, F8AMD) might be able to bind intercalatively to an RNA:DNA hybrid by forming an additional hydrogen bond between F8 and the 2'-hydroxyl group of the guanosine ribose. To test this hypothesis, the crystal structure of d(GAAGCTTC)2-F8AMD has been determined at 3.0 A resolution. Based on this crystal structure, a model in which F8AMD binds into the hybrid r(GAAGCUUC):d(GAAGCTTC) has been built using molecular mechanics and dynamic methods. These structural studies indicate that F8AMD binds intercalatively to a B-form double-stranded DNA whereas the drug intercalates into an RNA:DNA hybrid taking an A-form conformation. In the RNA:DNA hybrid complex, the F8 atom is located so as to be able to interact to an O2' hydroxyl group with either an O-H...F hydrogen bond or H+...F- electrostatic interaction. This interaction might stabilize the F8AMD molecule in the RNA:DNA hybrid. A binding study indicates that both actinomycin D (AMD) and F8AMD bind intercalatively not only to double-stranded DNAs, but also to RNA:DNA hybrids. Although the overall binding capacity of F8AMD (k = 4.5 x 10(5) M-1) is reduced slightly in comparison with AMD itself (k = 1.8 x 10(6) M-1), F8AMD tends to bind relatively more favorably than AMD to the RNA:DNA hybrids. The drugs' effects on RNA synthesis in HeLa cells indicates that the binding capacities of AMD and F8AMD correlates strongly to their RNA synthesis inhibitory activities. F8AMD required a concentration of 78 nM to inhibit RNA polymerase activity in HeLa cells by 50%, whereas AMD reached the same inhibitory level at 30 nM. Surprisingly, F8AMD exhibits unique selectivity against leukemia cells as does another C8-derivatized AMD analogue, N8AMD. F8AMD inhibits 50% of leukemia cell growth at less than 1.0 nM whereas 10- to 130-fold-higher drug concentrations are required to inhibit the growth of other tumor cell lines by 50%. The GI50 value of F8AMD for leukemia cells is the lowest among the GI50 values for all other AMD derivatives tested. By contrast, AMD is quite potent and kills most cells at less than 50 nM concentration, but it does not show any selectivity for certain cell lines. This indicates that AMD should have very limited use as an antitumor agent. It is difficult to rationalize why F8AMD and N8AMD show such strong selectivity against leukemia cells. However, this study and our previous study (J. Am. Chem. Soc. 1994, 116, 7971) indicated that F8AMD and N8AMD tended to bind more favorably to RNA:DNA hybrids. Thus, the unique antileukemia selectivity shown by F8AMD and N8AMD might be used by the agents binding to RNA:DNA hybrids rather than to double-stranded DNA.

Antineoplastic Agents↗

PubMed1996

Physical and biological characteristics of the antitumor drug actinomycin D analogues derivatized at N-methyl-L-valine residues.

The crystal structure of the DNA-actinomycin D (AMD) complex and a simple molecular modeling study indicated that AMD analogues derivatized at N-methyl-L-valine residues (fifth amino acid residue in the cyclic depsipeptide of AMD) could bind to DNA as strongly as the parent AMD. The analogues in which N-methyl-L-valine residues were replaced with L- and D-forms of N-methylvalines, N-methylthreonines, N-methylphenylalanies, N-methyltyrosines, and N-methyl-O-methyltyrosines have been totally synthesized. The characteristics of binding of the analogues to various DNAs including DNA-1 [d(TATATATGCATATATA)], DNA-2 [d(TATATACGCGTATATA)], DNA-3 [d(ATATATAGCTATATAT)], and DNA-4 [d(ATATATGGCCATATAT)] have been examined by using visible absorption spectrum methods. The association constants calculated from the absorption spectra indicate that the modifications of the N-methyl-L-valine residues in the AMD molecule do affect the DNA binding characteristics of the analogues. The L-aromatic analogues bind slightly better than the L-aliphatic analogues except for binding to DNA-1 (-TGCA-), whereas the D-aliphatic analogues bind consistently better than the D-aromatic analogues. In the L-form analogues, the L-Tyr analogue has the highest overall association constant, whereas the D-Val analogue has the highest association constant among the D-form analogues. In spite of substitution of bulky aromatic groups, the D-aromatic analogues bind to the DNA-1 quite well. However, D-aromatic analogues have significantly reduced their binding capacities to the other DNAs, indicating that the substitution of the D-aromatic residues creates a unique four-base sequence preference (-TGCA-). The RNA polymerase inhibitory activities of the AMD analogues in vivo have been examined using human cells (HeLa). All AMD analogues except for the L-Thr analogues severely inhibit RNA synthesis at relatively low drug concentrations. The D-Val, L-OMT, L-Phe, and D-Phe analogues inhibit RNA synthesis more strongly than the natural antibiotic (AMD itself).

Antibiotics, Antineoplastic↗

PubMed1996

Crystal structure of glycine N-methyltransferase from rat liver.

Glycine N-methyltransferase (GNMT) from rat liver is a tetrameric enzyme with 292 amino acid residues in each identical subunit and catalyzes the S-adenosylmethionine (AdoMet) dependent methylation of glycine to form sarcosine. The crystal structure of GNMT complexed with AdoMet and acetate, a competitive inhibitor of glycine, has been determined at 2.2 A resolution. The subunit of GNMT forms a spherical shape with an extended N-terminal region which corks the entrance of active site of the adjacent subunit. The active site is located in the near center of the spherical subunit. As a result, the AdoMet and acetate in the active site are completely surrounded by amino acid residues. Careful examination of the structure reveals several characteristics of GNMT. (1) Although the structure of the AdoMet binding domain of the GNMT is very similar to those of other methyltransferases recently determined by X-ray diffraction method, an additional domain found only in GNMT encloses the active site to form a molecular basket, and consequently the structure of GNMT looks quite different from those of other methyltransferases. (2) This unique molecular structure can explain why GNMT can capture folate and polycyclic aromatic hydrocarbons. (3) The unique N-terminal conformation and the subunit structure can explain why GNMT exhibits positive cooperativity in binding AdoMet. From the structural features of GNMT, we propose that the enzyme might be able to capture yet unidentified molecules in the cytosol and thus participates in various biological processes including detoxification of polycyclic aromatic hydrocarbons. In the active site, acetate binds near the S-CH3 moiety of AdoMet. Simple modeling indicates that the amino group of the substrate glycine can be placed close to the methyl group of AdoMet within 3.0 A and form a hydrogen bond with the carboxyl group of Glu15 of the adjacent subunit. On the basis of the ternary complex structure, the mechanism of the methyl transfer in GNMT has been proposed.

Amino Acid Sequence↗

PubMed1996

Structure and function of S-adenosylmethionine synthetase: crystal structures of S-adenosylmethionine synthetase with ADP, BrADP, and PPi at 28 angstroms resolution.

S-Adenosylmethionine synthetase (MAT,ATP:L-methionine S-adenosltransferase, EC 2.5.1.6) plays a central metabolic role in all organisms. MAT catalyzes the two-step reaction which synthesizes S-adenosylmethionine (AdoMet), pyrophosphate (PPi), and orthophosphate (Pi) from ATP and L-methionine. AdoMet is the primary methyl group donor in biological systems. The first crystal structure of MAT from Escherichia coli has recently been determined [Takusagawa et al. (1995) J. Biol. Chem. 271, 136-147]. In order to elucidate the active site and possible catalytic reaction mechanism, the MAT structures in the crystals grown with the substrate ATP (and BrATP) and the product PPi have been determined (space group P6(2)22; unit cell a = b = 128.9 Angstroms, c= 139.8 Angstroms, resolution limit 2.8 Angstroms; R O.19; Rfree 0.26). The enzyme consists of four identical subunits; two subunits form a spherical dimer, and pairs of these tightly bound dimers form a tetrameric enzyme. Each dimer has two active sites which are located between the subunits. Each subunit consists of three domains related to each other by a pseudo 3-fold symmetry. The crystal structures showed that the ATP molecules were hydrolyzed to ADP and Pi by the enzyme. Those products were found at the active site along with the essential metal ions (K+ and Mg2+). This rather unexpected finding was first confirmed by the structure of the complex with PPi and later by an HPLC analysis. The enzyme hydrolyzed ATP to ADP and Pi in 72 h under the same conditions as the crystallization of the enzyme. In the active site, the diphosphate moiety of ADP and Pi interacts extensively with amino acid residues from the two subunits of the enzyme, whereas the adenine and ribose moieties have little interaction with the enzyme. The enzyme structure is little changed upon binding ADP. All amino acid residues involved in the active site are found to be conserved in the 14 reported sequences of MAT from a wide range of organisms. Thus the structure determined in this study can be utilized as a model for other members of the MAT family. On the basis of the crystal structures, the catalytic reaction mechanisms of AdoMet formation and hydrolysis of tripolyphosphate are proposed.

Adenosine Diphosphate↗

PubMed1996

Crystal structure of S-adenosylmethionine synthetase.

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.

Amino Acid Sequence↗

PubMed1996

Flexible loop in the structure of S-adenosylmethionine synthetase crystallized in the tetragonal modification.

S-Adenosylmethionine synthetase (MAT, ATP:L-methionine S-adenosyltransferase, E.C.2.5.1.6.) plays a central metabolic role in all organisms. MAT catalyzes the two-step reaction which synthesizes S-adenosylmethionine (AdoMet), pyrophosphate (PPi) and orthophosphate (Pi) from ATP and L-methionine. AdoMet is the primary methyl group donor in biological systems. MAT from Escherichia coli was crystallized in the tetragonal modification with space group P4(3)2(1)2 using the same conditions as previously yielded crystals of the hexagonal system [Takusagawa, et al., (1996), J. Biol. Chem. 171, 136-147], except for the crystallization temperature. The structure has been determined by molecular replacement at 3.2 A resolution. The overall structure of the tetrameric MAT in the tetragonal modification is essentially the same as the structure found in the hexagonal modification. However there are two remarkable differences between the structures of two modifications. One is the contents in the active sites (holoform vs. apo-form), and the other is the conformation of the flexible loop over the active site (open vs. closed). These differences in the crystal structures are caused solely by the difference in crystallization temperatures (26 degrees C vs. 4 degrees C). We have interpreted the structural data obtained from the X-ray analyses in conjunction with the results of the mechanistic and sequencing studies in terms of possible dynamic motion of the flexible loop. When a substrate/product binds in the active site (hexagonal modification), the loop becomes disordered, apparently due to flexibility at the entrance of the active site as if it acts as a "mobile loop" during the catalytic reaction. On the other hand, when the temperature is decreased, the dynamic motion of the flexible loop may be reduced, and the loop residues enter the active site and close its entrance (tetragonal modification). Thus, the active site of the tetragonal modification is empty despite the crystals being grown in mother liquor containing a large concentration of phosphate (100 mM). There is no significant displacement of amino acid residues in the active site between the holo and apo forms, suggesting that the flexible loop plays an important role in determination of the contents in the active site. Since the functionally important amino acid residues in the active site are all conserved throughout various species, the structures of the active sites and the mechanism of the catalysis are probably essentially identical in the enzymes from a wide range of organisms. However, the substrate KM and Vmax values of MATs from various species are distributed over a wide range. The amino acid residues in the flexible loop regions are poorly conserved throughout various species. Therefore, the wide differences in catalysis rates of MATs from various speeches may be due to the differences in the composition of the flexible loop.

Amino Acid Sequence↗

PubMed1995

Structural, physical, and biological characteristics of RNA.DNA binding agent N8-actinomycin D.

The crystal structure of the self-complementary DNA octamer d(GAAGCTTC)2 complexed with N8-actinomycin D (N8AMD) has been determined at 3.0 A resolution (space group: P3(1)21; unit cell: a = 62.30, b = 62.30, c = 42.97 A; R = 0.173 for 1845 reflections). The DNA structure was severely distorted by the N8AMD bound intercalatively into the middle dinucleotide, 5'-GC-3'. The two cyclic depsipeptides, which differ from each other in overall conformation, lie in the minor groove. The complex is further stabilized by forming base--peptide and chromophore--backbone hydrogen bonds. The complexes are stacked together to form a pseudocontinuous helix running through the crystals. The structure of d(GAAGCTTC)2-actinomycin D (AMD) crystallized in the space group C2 [Kamitori S., & Takusagawa, F. (1992) J. Mol. Biol. 225, 445-456] was re-refined in order to compare it directly to the N8AMD complex structure. The asymmetrical binding mode of AMD has been confirmed on the basis of the two complex structures. The crystal structures of the N8AMD and AMD complexes bound to the same d(GAAGCTTC)2 differed by a root-mean-square deviation on all atom positions of 1.77 A, but most of the structural differences can be attributed to molecular packing in two different crystal forms, and not to structural differences induced by the interaction with the intercalating agents. However, the DNA binding and biological characteristics of N8AMD and AMD are quite different from each other. The DNA association constant of N8AMD is 33-fold less than that of AMD in an aqueous solution. N8AMD required a concentration > 10.0 microM to inhibit RNA synthesis activity in HeLa cells by 50%, whereas AMD reached to the same inhibitory level at only 35 nM. The structure of the DNA-N8AMD complex suggested that substitution of the N-methyl-L-valine residue in the cyclic depsipeptide with a N-methyl-D-valine residue might increase the hydrophobic interaction with the minor groove of the DNA. Thus the DNA association constant and RNA synthesis inhibitory activities of 5,5'-N-methyl-D-valine AMD (D-MeVal-AMD) have also been determined. The DNA association constant of D-MeVal-AMD is more than 2-fold greater than that of AMD, and the RNA synthesis inhibitory activity is about 20-fold greater.

Base Sequence↗

PubMed1993

Structure of the product from a novel cyclization reaction involving a C(6)-substituted uridine analog.

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.

Crystallography, X-Ray↗

PubMed1993

Characterization of the complexation of fluoroquinolone antimicrobials with metal ions by nuclear magnetic resonance spectroscopy.

The complexation of the fluoroquinolone antimicrobials is important because it has been implicated in reduced oral bioavailability and reduced antimicrobial activity when the drugs are co-administered with antacids or multi-vitamin preparations containing iron. The complexation of two model compounds, lomefloxacin and norflaxacin was studied using NMR. With aluminum ions, exchange between free and bound drug molecules was slow on the NMR time-scale. Two complexes, proposed to have stoichiometries of 2:1 and 3:1 (drug:metal) based on peak widths and variable temperature studies, were observed. The crystal structure of lomefloxacin, which shows intermolecular self association previously reported to be crucial to the drug's mode of action, is also reported. Because the metal ion complexes could not be crystallized, the crystal structure of uncomplexed lomefloxacin together with the NMR data on the aluminum complexes were used in the molecular modelling of the lomefloxacin-aluminum complexes.

Anti-Infective Agents↗