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J N Scarsdale

Publications and source records attributed to J N Scarsdale.

At least 19 recordsLinked to original sources
PubMed2001

Crystal structure of the Mycobacterium tuberculosis beta-ketoacyl-acyl carrier protein synthase III.

Mycolic acids (alpha-alkyl-beta-hydroxy long chain fatty acids) cover the surface of mycobacteria, and inhibition of their biosynthesis is an established mechanism of action for several key front-line anti-tuberculosis drugs. In mycobacteria, long chain acyl-CoA products (C(14)-C(26)) generated by a type I fatty-acid synthase can be used directly for the alpha-branch of mycolic acid or can be extended by a type II fatty-acid synthase to make the meromycolic acid (C(50)-C(56)))-derived component. An unusual Mycobacterium tuberculosis beta-ketoacyl-acyl carrier protein (ACP) synthase III (mtFabH) has been identified, purified, and shown to catalyze a Claisen-type condensation between long chain acyl-CoA substrates such as myristoyl-CoA (C(14)) and malonyl-ACP. This enzyme, presumed to play a key role in initiating meromycolic acid biosynthesis, was crystallized, and its structure was determined at 2.1-A resolution. The mtFabH homodimer is closely similar in topology and active-site structure to Escherichia coli FabH (ecFabH), with a CoA/malonyl-ACP-binding channel leading from the enzyme surface to the buried active-site cysteine residue. Unlike ecFabH, mtFabH contains a second hydrophobic channel leading from the active site. In the ecFabH structure, this channel is blocked by a phenylalanine residue, which constrains specificity to acetyl-CoA, whereas in mtFabH, this residue is a threonine, which permits binding of longer acyl chains. This same channel in mtFabH is capped by an alpha-helix formed adjacent to a 4-amino acid sequence insertion, which limits bound acyl chain length to 16 carbons. These observations offer a molecular basis for understanding the unusual substrate specificity of mtFabH and its probable role in regulating the biosynthesis of the two different length acyl chains required for generation of mycolic acids. This mtFabH presents a new target for structure-based design of novel antimycobacterial agents.

3-Oxoacyl-(Acyl-Carrier-Protein) Synthase↗
PubMed2000

Crystal structure at 2.4 A resolution of E. coli serine hydroxymethyltransferase in complex with glycine substrate and 5-formyl tetrahydrofolate.

Serine hydroxymethyltransferase (EC 2.1.2.1), a member of the alpha-class of pyridoxal phosphate enzymes, catalyzes the reversible interconversion of serine and glycine, changing the chemical bonding at the C(alpha)-C(beta) bond of the serine side-chain mediated by the pyridoxal phosphate cofactor. Scission of the C(alpha)-C(beta) bond of serine substrate produces a glycine product and most likely formaldehyde, which reacts without dissociation with tetrahydropteroylglutamate cofactor. Crystal structures of the human and rabbit cytosolic serine hydroxymethyltransferases (SHMT) confirmed their close similarity in tertiary and dimeric subunit structure to each other and to aspartate aminotransferase, the archetypal alpha-class pyridoxal 5'-phosphate enzyme. We describe here the structure at 2.4 A resolution of Escherichia coli serine hydroxymethyltransferase in ternary complex with glycine and 5-formyl tetrahydropteroylglutamate, refined to an R-factor value of 17.4 % and R(free) value of 19.6 %. This structure reveals the interactions of both cofactors and glycine substrate with the enzyme. Comparison with the E. coli aspartate aminotransferase structure shows the distinctions in sequence and structure which define the folate cofactor binding site in serine hydroxymethyltransferase and the differences in orientation of the amino terminal arm, the evolution of which was necessary for elaboration of the folate binding site. Comparison with the unliganded rabbit cytosolic serine hydroxymethyltransferase structure identifies changes in the conformation of the enzyme, similar to those observed in aspartate aminotransferase, that probably accompany the binding of substrate. The tetrameric quaternary structure of liganded E. coli serine hydroxymethyltransferase also differs in symmetry and relative disposition of the functional tight dimers from that of the unliganded eukaryotic enzymes. SHMT tetramers have surface charge distributions which suggest distinctions in folate binding between eukaryotic and E. coli enzymes. The structure of the E. coli ternary complex provides the basis for a thorough investigation of its mechanism through characterization and structure determination of site mutants.

Amino Acid Sequence↗
PubMed1999

Crystal structure of rabbit cytosolic serine hydroxymethyltransferase at 2.8 A resolution: mechanistic implications.

Serine hydroxymethyltransferase (SHMT) catalyzes the reversible cleavage of serine to form glycine and single carbon groups that are essential for many biosynthetic pathways. SHMT requires both pyridoxal phosphate (PLP) and tetrahydropteroylpolyglutamate (H4PteGlun) as cofactors, the latter as a carrier of the single carbon group. We describe here the crystal structure at 2.8 A resolution of rabbit cytosolic SHMT (rcSHMT) in two forms: one with the PLP covalently bound as an aldimine to the Nepsilon-amino group of the active site lysine and the other with the aldimine reduced to a secondary amine. The rcSHMT structure closely resembles the structure of human SHMT, confirming its similarity to the alpha-class of PLP enzymes. The structures reported here further permit identification of changes in the PLP group that accompany formation of the geminal diamine complex, the first intermediate in the reaction pathway. On the basis of the current mechanism derived from solution studies and the properties of site mutants, we are able to model the binding of both the serine substrate and the H4PteGlun cofactor. This model explains the properties of several site mutants of SHMT and offers testable hypotheses for a more detailed mechanism of this enzyme.

Amino Acid Sequence↗
PubMed1998

Identification and hydropathic characterization of structural features affecting sequence specificity for doxorubicin intercalation into DNA double-stranded polynucleotides.

The computer molecular modeling program HINT (Hydropathic INTeractions), an empirical hydropathic force field function that includes hydrogen bonding, coulombic and hydrophobic terms, was used to study sequence-selective doxorubicin binding/intercalation in the 64 unique CAxy, CGxy, TAxy, TGxy base pair quartet combinations. The CAAT quartet sequence is shown to have the highest binding score of the 64 combinations. Of the two regularly alternating polynucleotides, d(CGCGCG)2and d(TATATA)2, the HINT calculated binding scores reveal doxorubicin binds preferentially to d(TATATA)2. Although interactions of the chromophore with the DNA base pairs defining the intercalation site [I-1] [I+1] and the neighboring [I+2] base pair are predominant, the results obtained with HINT indicate that the base pair [I+3] contributes significantly to the sequence selectivity of doxorubicin by providing an additional hydrogen bonding opportunity for the N3' ammonium of the daunosamine sugar moiety in approximately 25% of the sequences. This observation, that interactions involving a base pair [I+3] distal to the intercalation site play a significant role in stabilizing/destabilizing the intercalation of doxorubicin into the various DNA sequences, has not been previously reported. In general terms, this work shows that molecular modeling and careful analysis of molecular interactions can have a significant role in designing and evaluating nucleotides and antineoplastic agents.

Base Pairing↗
PubMed1995

Accumulation of isogloboside and ganglio-N-tetraosyl ceramide having blood group B determinant in the hepatomas of female LEC rats.

We have studied the neutral glycolipid composition of spontaneous hepatomas in LEC female rats. Neutral lipid fractions were isolated and purified by column chromatographies on DEAE-Toyopearl 650(M) and Iatrobeads. The neutral glycolipid fraction contained 3.2 to 4.4 micrograms lipid-bound glucose (Glc) per mg protein, and consisted of isogloboside (iso-Gb4, 50.8% of total neutral glycolipids) and IV3Gal, IV2Fuc, GgOse4Cer (asialo-BGM1, 13.5%) as the major neutral glycolipids and Gb3 and iso-Gb3 (9.2%), GlcCer (7.2%), LacCer (6.1%) as the other species. The structure of iso-Gb4 was elucidated by gas-liquid chromatography (GLC), permethylation study, liquid secondary ion (LSI) mass spectrometry, and nuclear Overhauser enhancement spectroscopy (NOESY) and that for asialo-BGM1 by GLC, LSI mass spectrometry, and high-performance thin-layer chromatography (HPTLC)-overlay method using anti-asialo-BGM1 antibody. Isogloboside and asialo-BGM1 which are found in negligible amounts in normal liver tissues may represent excellent markers for studying tumor metastasis and cellular adhesion.

ABO Blood-Group System↗
PubMed1995

Structural basis for serpin inhibitor activity.

The mechanism of formation and the structures of serpin-inhibitor complexes are not completely understood, despite detailed knowledge of the structures of a number of cleaved and uncleaved inhibitor, noninhibitor, and latent serpins. It has been proposed from comparison of inhibitor and noninhibitor serpins in the cleaved and uncleaved forms that insertion of strand s4A into preexisting beta-sheet A is a requirement for serpin inhibitor activity. We have investigated the role of this strand in formation of serpin-proteinase complexes and in serpin inhibitor activity through homology modeling of wild type inhibitor, mutant substrate, and latent serpins, and of putative serpin-proteinase complexes. These models explain the high stability of the complexes and provide an understanding of substrate behavior in serpins with point mutations in s4A and of latency in plasminogen activator inhibitor I.

Amino Acid Sequence↗
PubMed1993

Characterization of a hamster melanoma-associated ganglioside antigen as 7-O-acetylated disialoganglioside GD3.

We previously reported a hamster animal model of melanoma in which the tumor tissue expresses gangliosides GM3, GD3, and O-acetyl GD3. This ganglioside pattern is similar to that in human melanomas (Ren, S., A. Slominski, and R. K. Yu. 1989 Cancer Res. 49: 7051). In this study, we isolated and purified these gangliosides using chloroform-methanol extraction, Folch partition, chromatographies on DEAE-Sephadex A-25, and Iatrobeads columns. The yields of gangliosides GM3, GD3, and O-acetyl GD3 were 44.1 mg, 19.6 mg, and 9 mg per 100 g of Ma melanotic melanoma tissues, respectively. The structures of these gangliosides were characterized by periodate oxidation, gas chromatographic (GC) analysis, fast-atom bombardment-mass spectrometry (FAB-MS), and nuclear magnetic resonance (NMR) studies. The structure of hamster melanoma O-acetyl GD3 is different from the 9-O-acetyl GD3 previously reported in human melanoma. The major fatty acids of this ganglioside are C16:0, C18:0, C20:0, C22:0, and C24:0 and the long-chain base is C18-sphingosine.

Animals↗
PubMed1992

O-acetylated gangliosides in bovine buttermilk. Characterization of 7-O-acetyl, 9-O-acetyl, and 7,9-di-O-acetyl GD3.

Three O-acetylated gangliosides, G1, G2, and G3, were purified from bovine buttermilk by using chloroform/methanol extraction, Folch partitioning, chromatography on DEAE-Sephadex A-25, and Iatrobeads columns. The final yields of gangliosides G1, G2, and G3 were 2 mg, 37 mg, and 40 mg per 1.7 kg of the buttermilk powder, respectively. On the basis of immunostaining on high performance thin layer chromatography with specific monoclonal antibodies, mild alkaline treatment, gas-liquid chromatographic analysis, fast atom bombardment mass spectrometry, and proton nuclear magnetic resonance studies, G1 and G2 are characterized as O-acetylated GD3 and G3 as O-acetylated GT3, and the structures of these gangliosides are as follows: [formula: see text] The major fatty acids of these gangliosides were C18:0, C22:0, C23:0, and C24:0, and the long chain base was C18-sphingosine.

Acetylation↗
PubMed1991

Calorimetric and spectroscopic examination of the solution phase structures of prekallikrein binding domain peptides of high molecular weight kininogen.

Unique sequence-binding sites are exposed on the surface of high molecular weight kininogen which complex prekallikrein or factor XI with high affinity and specificity. A sequence comprising 31 residues of the mature kininogen molecule (Asp565-Lys595) retains full binding activity for prekallikrein (KD = 20 nM) and assumes a complex folded structure in solution which is stabilized by long-range interactions between N- and C-terminal residues. The sequence Trp569-Lys595 (27 residues) shows only 28% of this binding affinity and lacks the key structural features required for protein recognition (Scarsdale, J. N., and Harris, R. B., J. Prot. Chem. 9, 647-659, 1990). We were thus able to predict that N- or C-terminal truncations of the binding-site sequence would disrupt the conformational integrity required for binding. Two new peptides of 20- and 22- residues have now been synthesized and their solution phase structures examined. These peptides are N- and C-terminal truncations, respectively, of the 27-residue sequence and correspond to the sequences Asp576-Lys595 and Trp569-Asp590 of high molecular weight kininogen. The results of fluorescence emission and circular dichroism (CD) spectroscopies in the range 25-90 degrees C and from differential scanning calorimetry (DSC) all substantiate the idea that the C-terminal truncation peptide binds prekallikrein 35-fold poorer than the 31-residue peptide because it is relatively unordered and possesses a less stable structure. Surprisingly, the N-terminal truncation peptide (20-mer) shows structural stability even at elevated temperatures and, like the 31-residue peptide, undergoes cold-induced denaturation observable in the DSC. 2D-NMR analysis of the 20-residue peptide revealed two distinct structures; one conformer possesses a more compact, folded structure than the other. However, the predicted structures assumed by either conformer are very different from those of either the 31- or 27-residue peptides. Hence, the binding affinity of the 20-residue peptide is 60-fold poorer than that for the 31-residue peptide because it assumes a nonproductive binding conformation(s).

Amino Acid Sequence↗
PubMed1990

NMR and computational studies of interactions between remote residues in gangliosides.

Conformational preferences of the gangliosides GM1, GM1b, and GD1a have been investigated by using a systematic combination of NMR distance constraints and molecular mechanics calculations. These gangliosides share a common four-sugar core but differ in the number or placement of sialic acid residues attached to the core. Placement of the sialic acid residues is shown to influence the preferred core conformation. The origin of these effects is postulated to be intramolecular interactions of the sialic acid residues with other remote residues. In the case of GM1, hydrogen bonds between the internal sialic acid and an N-acetyl group on GalNAc are suggested. In the case of GD1a, a hydrogen-bonding network between the terminal and internal sialic acids is suggested to play a role.

Alcohol Oxidoreductases↗
PubMed1990

Solution phase conformation studies of the prekallikrein binding domain of high molecular weight kininogen.

High molecular weight kininogen is a cofactor of the surface-dependent phase of the blood-clotting cascade. Unique sequence-binding sites are exposed on the surface of this glycoprotein which complex prekallikrein or factor XI with high affinity and specificity (Tait and Fujikawa, 1987). A sequence comprising 31-residues (residues 565-595 of the mature kininogen molecule) retains full binding activity for prekallikrein but the sequence 569-595 (27 residues) shows only 25% of this binding affinity (Vogel et al., 1990). Thus, the key structural features required for protein recognition reside in the 31-residue sequence but these features are likely compromised (or absent) in the 27-residue sequence. To determine the conformation of the prekallikrein-binding domain, peptides comprising the 31- and 27-residue sequences were prepared by solid-phase methods and their structures determined by circular dichroism, fluorescence polarization, and 2D-NMR techniques. Fluorescence emission spectra, polarization, and anisotropy measurements of the single Trp residue present in both peptides show that the 31-residue peptide contains an ordered microenvironment at its amino terminus, which is not present in the 27-residue peptide. This structural ordering is characterized by movement of the Trp residue into a more polar environment. Further, the 31-residue peptide possesses a higher limit anisotropy, longer rotational relaxation time, and shows a higher polarization value even at elevated temperatures. Circular dichroic spectra of both peptides in the far UV region are essentially identical and indicate that both peptides contain predominantly beta-turn elements, but also contain some alpha-helix, beta-sheet, and random coil character. The structural elements of both peptides are unchanged in urea solution, but the negative ellipticity absorption band in the near UV region assignable to Trp is eliminated in acid solution upon protonation of the neighboring-Asp-Asp-Asp- triplet. In the two peptides, the spin system of each amino acid has been assigned through 2D-1H scalar coupling correlated experiments; pure absorption NOESY experiments were used to determine through-space connectivities. The results are entirely consistent with the previous experiments in that both peptides contain predominantly beta-turn elements and the amino terminus of the 31-residue peptide is highly ordered in comparison with the 27-mer; in fact, this region is likely to be helical in nature.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1989

High resolution proton NMR studies of gangliosides. Structure of two types of GD3 lactones and their reactivity with monoclonal antibody R24.

Ganglioside GD3 was converted at room temperature to two stable lactones, denoted as GD3 lactones I and II. The reaction sequence was presumed to be GD3----GD3 lactone I----GD3 lactone II based on the time course of their production. Lactone I behaved as a monosialoganglioside and lactone II as a neutral species. The two lactones were isolated by DEAE-Sephadex column chromatography. The positions of the inner ester linkages were investigated by two-dimensional J-correlated proton NMR spectroscopy. An ester linkage was most likely formed between the carboxyl group of the external sialic acid residue and C9-OH of the internal sialic acid residue in lactone I. In addition to this ester linkage, a second ester linkage between the carboxyl group of the internal sialic acid and C2-OH of the galactose residue was likely formed in lactone II. The structural changes induced by lactonization were further examined by their reactivity with the monoclonal antibody R24 (Puckel, C. S., Lloyd, K. O., Travassos, L. R., Dippold, W. G., Oettgen, H. F., and Old, L. J. (1982) J. Exp. Med. 155, 1133-1147), which reacted with GD3. R24 was found to bind weakly to GD3 lactone I, but not to GD3 lactone II. The results suggest that the monoclonal antibody requires both sialic acid residues for high affinity binding, and the complete lactonization results in a loss of negative charges and/or a change in the overall conformation of the oligosaccharide moiety which may account for the loss of binding.

Animals↗
PubMed1988

Accumulation of a globo-series glycolipid having Gal alpha 1-3Gal in PC12h pheochromocytoma cells.

In a previous paper, we reported the presence of globoside as a major neutral glycolipid in PC12 pheochromocytoma cells [Ariga, T., Macala, L. J., Saito, M., Margolis, R. K., Greene, L. A., Margolis, R. U., & Yu, R. K. (1988) Biochemistry 27, 52-58]. Recently, we found that subcloned PC12h cells accumulated another unusual neutral glycolipid. In order to characterize this glycolipid, PC12h cells were subcutaneously transplanted into rats. The induced tumor tissue accumulated two major neutral glycolipids, which were purified by Iatrobeads column and preparative thin-layer chromatographies. One of the glycolipids was found to be globoside, and the other had a globotriaosyl structure with an additional terminal Gal alpha 1-3 residue. Its structure was determined by fast atom bombardment mass spectrometry, two-dimensional proton nuclear magnetic resonance spectrometry (2D NMR), permethylation study, sequential degradation with exoglycosidase, and mild acid hydrolysis to be Gal(alpha 1-3)Gal(alpha 1-4)Gal(beta 1-4)Glc(beta 1-1')Cer.

Adrenal Gland Neoplasms↗
PubMed1986

Elucidation of glycolipid structure by proton nuclear magnetic resonance spectroscopy.

The primary structure of the oligosaccharide moiety of a glycosphingolipid can be elucidated by employing high-field proton nuclear magnetic resonance (NMR) spectroscopy. Information with respect to the composition and configuration of its sugar residues, and the sequence and linkage sites of the oligosaccharide chain can be obtained by employing a variety of one- and two-dimensional techniques. The latter include both scalar and dipolar correlated two-dimensional NMR spectroscopy. These techniques are also useful in establishing the solution conformation (secondary structure) of the oligosaccharide moiety. Examples in utilizing these techniques in elucidating the primary and secondary structures of glycolipids are presented.

Carbohydrate Conformation↗
PubMed1986

1H-2D-nuclear magnetic resonance applied to the primary structure determination of a novel octasaccharide glycolipid isolated from the spermatozoa of bivalves.

High resolution, two-dimensional 1H-n.m.r. spectroscopy has been used to confirm a proposed primary structure of a glycolipid having an octasaccharide head-group. Pure absorption and relay experiments were found to be particularly useful in establishing connectivities in poorly resolved regions of the spectrum. The spectral assignments, which indicate novel linkages including an internal fucopyranosyl residue as well as terminal xylosyl and 4-O-methylglucopyranosyluronic acid groups, add to a growing data base for structural characterization through n.m.r. spectroscopy.

Animals↗
PubMed1984

Recent advances in structural analysis of gangliosides: primary and secondary structures.

High-field (500 MHz) proton NMR has been used to elucidate the primary and secondary structures of glycosphingolipids (GSLs). Using 2-D J-correlated spectroscopy (2-D SECSY) which establishes scalar couplings of protons, the monosaccharide composition, anomeric configuration and aglycon structures of a GSL can be established. 2-D nuclear Overhauser effect spectroscopy (2-D NOE) then establishes through-space intra- and inter-residue couplings of cross-relaxing protons. We have found that each anomeric proton is involved in NOE couplings with inter- and intra-residue protons. The inter-residue coupling, resulting from interaction of protons across the glycosidic linkage, establishes the n-1 sugar residue and specific glycosidation site to which the n-residue is linked. When such information is known for each residue and is combined, the sequence of the core oligosaccharide is obtained. The sialylation-induced glycosidation shift is then used to establish the site of sialic acid residue attachment in a ganglioside molecule. We have also observed that the anomeric proton inter-residue NOE couplings can be used to suggest the preferred conformation of an oligosaccharide. We have found that the oligosaccharide residue of globoside exists in a unique and rather rigid conformation which could be stabilized by hydrogen bonds and van der Waals interactions. Since GSLs are known to have a receptor role and are implicated in cell-cell recognition, enzyme-substrate interaction and antigen-antibody interaction, the determination of their conformation should be useful in understanding their biological functions.

Animals↗
PubMed1984

Ab initio study on the molecular structure of the naphthalene metabolite, trans-1,2-dihydroxy-1,2-dihydronaphthalene.

The molecular geometries of three conformations of trans-1,2-dihydroxy-1,2-dihydronaphthalene have been refined by an ab initio gradient procedure at the 4-21G level to determine the effect of dihydrodiol conformation on arene structure. The preferred conformation is an equatorial form similar to the most stable conformation of ethylene glycol. All the structures investigated have similar arene geometries. The effect of the various conformations on metabolism of dihydrodiols to dihydrodiol epoxides is considered.

Models, Molecular↗