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J C Sacchettini

Publications and source records attributed to J C Sacchettini.

At least 55 records · Page 3Linked to original sources
PubMed1997

X-ray crystallographic studies of unique cross-linked lattices between four isomeric biantennary oligosaccharides and soybean agglutinin.

Soybean agglutinin (SBA) (Glycine max) is a tetrameric GalNAc/Gal-specific lectin which forms unique cross-linked complexes with a series of naturally occurring and synthetic multiantennary carbohydrates with terminal GalNAc or Gal residues [Gupta et al. (1994) Biochemistry 33, 7495-7504]. We recently reported the X-ray crystal structure of SBA cross-linked with a biantennary analog of the blood group I carbohydrate antigen [Dessen et al. (1995) Biochemistry 34, 4933-4942]. In order to determine the molecular basis of different carbohydrate-lectin cross-linked lattices, a comparison has been made of the X-ray crystallographic structures of SBA cross-linked with four isomeric analogs of the biantennary blood group I carbohydrate antigen. The four pentasaccharides possess the common structure of (beta-LacNAc)2Gal-beta-R, where R is -O(CH2)5COOCH3. The beta-LacNAc moieties in the four carbohydrates are linked to the 2,3-, 2,4-, 3,6-, and 2,6-positions of the core Gal residue(s), respectively. The structures of all four complexes have been refined to approximately 2.4-2.8 A. Noncovalent lattice formation in all four complexes is promoted uniquely by the bridging action of the two arms of each bivalent carbohydrate. Association between SBA tetramers involves binding of the terminal Gal residues of the pentasaccharides at identical sites in each monomer, with the sugar(s) cross-linking to a symmetry-related neighbor molecule. While the 2,4-, 3,6-, and 2,6-pentasaccharide complexes possess a common P6422 space group, their unit cell dimensions differ. The 2, 3-pentasaccharide cross-linked complex, on the other hand, possesses the space group I4122. Thus, all four complexes are crystallographically distinct. The four cross-linking carbohydrates are in similar conformations, possessing a pseudo-2-fold axis of symmetry which lies on a crystallographic 2-fold axis of symmetry in each lattice. In the case of the 3,6- and 2,6-pentasaccharides, the symmetry of their cross-linked lattices requires different rotamer orientations about their beta(1,6) glycosidic bonds. The results demonstrate that crystal packing interactions are the molecular basis for the formation of distinct cross-linked lattices between SBA and four isomeric pentasaccharides. The present findings are discussed in terms of lectins forming unique cross-linked complexes with glycoconjugate receptors in biological systems.

Carbohydrate Conformation↗
PubMed1997

The three-dimensional structure of an H-2Ld-peptide complex explains the unique interaction of Ld with beta-2 microglobulin and peptide.

Solution at 2.5-A resolution of the three-dimensional structure of H-2Ld with a single nine-residue peptide provides a structural basis for understanding its unique interaction with beta-2 microglobulin (beta2m) and peptide. Consistent with the biological data that show an unusually weak association of Ld with beta2m, a novel orientation of the alpha1/alpha2 domains of Ld relative to beta2m results in a dearth of productive contacts compared with other class I proteins. Characteristics of the Ld antigen-binding cleft determine the unique motif of peptides that it binds. Ld has no central anchor residue due to the presence of several bulky side chains in its mid-cleft region. Also, its cleft is significantly more hydrophobic than that of the other class I molecules for which structures are known, resulting in many fewer H-bonds between peptide and cleft residues. The choice of Pro as a consensus anchor at peptide position 2 appears to be related to the hydrophobicity of the B pocket, and to the unique occurrence of Ile (which mirrors Pro in its inability to form H-bonds) at position 63 on the edge of this pocket. Thus, the paucity of stabilizing H-bonds combined with poor complementarity between peptide postion 2 Pro and the B pocket contribute to the weak association between Ld and its peptide antigen. The unique structural interactions of Ld with beta2m and peptide could make Ld more suited than other classical class I molecules to play a role in alternative pathways of antigen presentation.

Animals↗
PubMed1997

Kinetic mechanism of human hypoxanthine-guanine phosphoribosyltransferase: rapid phosphoribosyl transfer chemistry.

Hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) is the locus of Lesch-Nyhan syndrome, the activator of the prodrugs 6-mercaptopurine and allopurinol, and a target for antiparasitic chemotherapy. The three-dimensional structure of the recombinant human enzyme in complex with GMP has recently been solved [Eads, J., Scapin, G., Xu, Y., Grubmeyer, C., & Sacchettini, J. C. (1994) Cell 78, 325-334]. Here, ligand binding, pre-steady state kinetics, isotope trapping, and isotope exchange experiments are presented which detail the sequential kinetic mechanism of the enzyme. In the forward reaction, in which a base (hypoxanthine or guanine) reacts with PRPP to form nucleoside monophosphate and PPi, binding of PRPP precedes that of the base, and in the reverse direction, IMP binds first. Compared to k(cat), phosphoribosyl group transfer is rapid in both the forward (131 vs 6.0 s(-1)) and reverse (9 vs 0.17 s(-1)) directions. In the forward direction, product pyrophosphate dissociates rapidly (> 12 s(-1)) followed by release of IMP (6.0 s(-1)). In the reverse direction, Hx dissociates rapidly (9.5 s(-1)) and PRPP dissociates slowly (0.24 s(-1)). The more rapid rate of utilization of guanine than hypoxanthine in the forward reaction is the result of the faster release of product GMP rather than the result of differences in the rate of the chemical step. The kinetic mechanism, with rapid chemistry and slow product dissociation, accounts for the previously observed ability of the alternative product guanine to stimulate, rather than inhibit, the pyrophosphorolysis of IMP. The overall equilibrium for the hypoxanthine phosphoribosyl transfer reaction lies far toward nucleotide product (Keq approximately 1.6 x 10(5)), at the high end for PRPP-linked nucleotide formation. The three-dimensional structure of the HGPRTase x IMP complex has been solved to 2.4 A resolution and is isomorphous with the GMP complex. The results of the ligand binding and kinetic studies are discussed in light of the structural data.

Guanine↗
PubMed1997

A new function for a common fold: the crystal structure of quinolinic acid phosphoribosyltransferase.

BACKGROUND: Quinolinic acid (QA) is a neurotoxin and has been shown to be present at high levels in the central nervous system of patients with certain diseases, such as AIDS and meningitis. The enzyme quinolinic acid phosphoribosyltransferase (QAPRTase) provides the only route for QA metabolism and is also an essential step in de novo NAD biosynthesis. QAPRTase catalyzes the synthesis of nicotinic acid mononucleotide (NAMN) from QA and 5-phosphoribosyl-1-pyrophosphate (PRPP). The structures of several phosphoribosyltransferases (PRTases) have been reported, and all have shown a similar fold of a five-strandard beta sheet surrounded by four alpha helices. A conserved sequence motif of 13 residues is common to these 'type I' PRTases but is not observed in the QAPRTase sequence, suggestive of a different fold for this enzyme. RESULTS: The crystal structure of QAPRTase from Salmonella typhimurium has been determined with bound QA to 2.8 A resolution, and with bound NAMN to 3.0 A resolution. Most significantly, the enzyme shows a completely novel fold for a PRTase enzyme comprising a two-domain structure: a mixed alpha/beta N-terminal domain and an alpha/beta barrel-like domain containing seven beta strands. The active site is located at the C-terminal ends of the beta strands of the alpha/beta barrel, and is bordered by the N-terminal domain of the second subunit of the dimer. The active site is largely composed of a number of conserved charged residues that appear to be important for substrate binding and catalysis. CONCLUSIONS: The seven-stranded alpha/beta-barrel domain of QAPRTase is very similar in structure to the eight-stranded alpha/beta-barrel enzymes. The structure shows a phosphate-binding site that appears to be conserved among many alpha/beta-barrel enzymes including indole-3-glycerol phosphate synthase and flavocytochrome b2. The new fold observed here demonstrates that the PRTase enzymes have evolved their similar chemistry from at least two completely different protein architectures.

Amino Acid Sequence↗
PubMed1997

Solution structure of human intestinal fatty acid binding protein: implications for ligand entry and exit.

The human intestinal fatty acid binding protein (I-FABP) is a small (131 amino acids) protein which binds dietary long-chain fatty acids in the cytosol of enterocytes. Recently, an alanine to threonine substitution at position 54 in I-FABP has been identified which affects fatty acid binding and transport, and is associated with the development of insulin resistance in several populations including Mexican-Americans and Pima Indians. To investigate the molecular basis of the binding properties of I-FABP, the 3D solution structure of the more common form of human I-FABP (Ala54) was studied by multidimensional NMR spectroscopy. Recombinant I-FABP was expressed from E. coli in the presence and absence of 15N-enriched media. The sequential assignments for non-delipidated I-FABP were completed by using 2D homonuclear spectra (COSY, TOCSY and NOESY) and 3D heteronuclear spectra (NOESY-HMQC and TOCSY-HMQC). The tertiary structure of human I-FABP was calculated by using the distance geometry program DIANA based on 2519 distance constraints obtained from the NMR data. Subsequent energy minimization was carried out by using the program SYBYL in the presence of distance constraints. The conformation of human I-FABP consists of 10 antiparallel beta-strands which form two nearly orthogonal beta-sheets of five strands each, and two short alpha-helices that connect the beta-strands A and B. The interior of the protein consists of a water-filled cavity between the two beta-sheets. The NMR solution structure of human I-FABP is similar to the crystal structure of rat I-FABP. The NMR results show significant conformational variability of certain backbone segments around the postulated portal region for the entry and exit of fatty acid ligand.

Amino Acid Sequence↗
PubMed1996

Crystallization of a deglycosylated T cell receptor (TCR) complexed with an anti-TCR Fab fragment.

A strategy to overexpress T cell receptors (TCRs) in Lec3.2.8.1 cells has been developed using the "Velcro" leucine zipper sequence to facilitate alpha-beta pairing. Upon secretion in culture media, the VSV-8-specific/H2-Kb-restricted N15 TCR could be readily immunopurified using the anti-leucine zipper monoclonal antibody 2H11, with a yield of 5-10 mg/liter. Mass spectrometry analysis revealed that all attached glycans were GlcNAc2-Man5. Following Superdex 200 gel filtration to remove aggregates, wild-type N15 or N15(s), a C183S variant lacking the unpaired cysteine at amino acid residue 183 in the Cbeta domain, was thrombin-cleaved and endoglycosidase H-digested, and the two derivatives were termed iN15DeltaH and N15(s)DeltaH, respectively, and sized by Superdex 75 chromatography to high purity. N-terminal and C-terminal microsequencing analysis showed the expected unique termini of N15 alpha and beta subunits. Nevertheless, neither protein crystallized under a wide range of conditions. Subsequently, we produced a Fab fragment of the murine TCR Cbeta-specific hamster monoclonal antibody H57 and complexed the Fab fragment with iN15DeltaH and N15(s)DeltaH. Both N15(s)DeltaH-Fab[H57] and iN15DeltaH-Fab[H57] complexes crystallize, with the former diffracting to 2.8-A resolution. These findings show that neither intact glycans nor the conserved and partially exposed Cys-183 is required for protein stability. Furthermore, our results suggest that the H57 Fab fragment aids in the crystallization of TCRs by altering their molecular surface and/or stabilizing inherent conformational mobility.

Animals↗
PubMed1996

Regulation of product chain length by isoprenyl diphosphate synthases.

An analysis of the x-ray structure of homodimeric avian farnesyl diphosphate synthase (geranyltransferase, EC 2.5.1.10) coupled with information about conserved amino acids obtained from a sequence alignment of 35 isoprenyl diphosphate synthases that synthesize farnesyl (C15), geranylgeranyl (C20), and higher chain length isoprenoid diphosphates suggested that the side chains of residues corresponding to F112 and F113 in the avian enzyme were important for determining the ultimate length of the hydrocarbon chains. This hypothesis was supported by site-directed mutagenesis to transform wild-type avian farnesyl diphosphate synthase (FPS) into synthases capable of producing geranylgeranyl diphosphate (F112A), geranylfarnesyl (C25) diphosphate (F113S), and longer chain prenyl diphosphates (F112A/F113S). An x-ray analysis of the structure of the F112A/F113S mutant in the apo state and with allylic substrates bound produced the strongest evidence that these mutations caused the observed change in product specificity by directly altering the size of the binding pocket for the growing isoprenoid chain in the active site of the enzyme. The proposed binding pocket in the apo mutant structure was increased in depth by 5.8 A as compared with that for the wild-type enzyme. Allylic diphosphates were observed in the holo structures, bound through magnesium ions to the aspartates of the first of two conserved aspartate-rich sequences (D117-D121), with the hydrocarbon tails of all the ligands growing down the hydrophobic pocket toward the mutation site. A model was constructed to show how the growth of a long chain prenyl product may proceed by creation of a hydrophobic passageway from the FPS active site to the outside surface of the enzyme.

Alkyl and Aryl Transferases↗
PubMed1996

Flexibility is a likely determinant of binding specificity in the case of ileal lipid binding protein.

BACKGROUND: The family of lipid binding proteins (LBPs) includes a large number of fatty acid binding proteins (FABPs) but only two proteins (ileal lipid binding protein, ILBP, and liver fatty acid binding protein) that can bind both fatty acids and bile acids. Bile acid transport is medically and pharmacologically important, but is poorly understood. To understand the binding properties of ILBP, we studied its solution structure with and without bound lipids and compared these with known structures of FABPs. RESULTS: The sequence-specific 1H resonance assignments for porcine ILBP have been determined by homonuclear two-dimensional (2D) NMR spectroscopy for the apo-protein as well as for ILBP complexes with fatty acid and bile acid ligands. From NOE spectra and hydrogen exchange data, similar secondary structure elements were identified for all three protein forms. ILBP is composed of ten antiparallel beta strands arranged in two nearly orthogonal beta sheets (a fold seen in other FABPs, and dubbed the "beta-clam shell'), covered on one side by two short, nearly parallel alpha helices. Binding of fatty acids or bile acids to ILBP alters mainly the side-chain proton resonances of amino acids within the protein cavity, indicating that both bile acids and fatty acids can bind in the interior of the protein between the two beta sheets; binding of bile acids stabilizes the protein backbone by a small amount. Fast hydrogen exchange rates for the backbone amide protons of ILBP indicate that the hydrogen-bonding network of the beta sheet in ILBP is weaker than the corresponding network in rat intestinal and bovine heart FABPs. CONCLUSIONS: The tertiary structure of ILBP is similar to that of other LBPs, but appears to be unusually flexible, with a relatively weak hydrogen-bonding network. It is likely that this flexibility is important in allowing bile acids, which are larger and more rigid than fatty acids, to enter the central cavity of the protein.

Amino Acid Sequence↗
PubMed1996

Inosine-uridine nucleoside hydrolase from Crithidia fasciculata. Genetic characterization, crystallization, and identification of histidine 241 as a catalytic site residue.

Protozoa depend on purine salvage for nucleic acid synthesis. An abundant salvage enzyme in Crithidia fasciculata is the inosine-uridine nucleoside hydrolase (IU-nucleoside hydrolase). The enzyme was cloned by polymerase chain reaction techniques using primers corresponding to the amino acid sequences of tryptic fragments and to the miniexon of C. fasciculata. The full-length cDNA was expressed in Escherichia coli and the protein purified to > 99% homogeneity. The open reading frame encodes a protein of 315 amino acids. Enzyme purified from C. fasciculata was missing the N-terminal Met and gave a major mass peak of 34 194 amu by mass spectrometry. Predicted mass from the DNA sequence for the Met-processed enzyme was 34 196. A pET3d-IUNH construct expressed in E. coli introduced MetAla instead of MetPro at the N-terminus. Enzyme purified from this construct also had a processed N-terminus and gave predicted and observed masses of 34 168 and 34 170 amu, respectively. The amino acid sequence for IU-nucleoside hydrolase has no close relatives among the known proteins. A cDNA clone of unknown function from Leishmania major shows near identity in the N-terminal deduced amino acid sequence. Open reading frames near 1 and 47 min on the E. coli chromosome and from two yeast genomes encode for proteins of similar size with substantial amino acid identity. Mutation of His241Ala caused a 2100-fold loss in k(cat) for inosine but a 2.8-fold increase in k(cat) with p-nitrophenyl beta-D-ribofuranoside, establishing the location of the catalytic site and implicating His241 as a proton donor for leaving group activation. IU-nucleoside hydrolase from C. fasciculata and the protein expressed in E. coli were crystallized and diffract to 2.5 and 2.1 A resolution, respectively. Both belong to the P2(1)2(1)2 orthorhombic space group with unit cell parameters a = 63.5 A, b = 131.9 A, c = 90.1 A, and alpha = beta = gamma = 90 degrees. Two subunits of the tetrameric enzyme are present in the asymmetric unit. The following paper reports the X-ray crystal structure for this enzyme.

Amino Acid Sequence↗
PubMed1996

Three-dimensional structure of the inosine-uridine nucleoside N-ribohydrolase from Crithidia fasciculata.

Protozoan parasites rely on the host for purines since they lack a de novo synthetic pathway. Crithidia fasciculata salvages exogenous inosine primarily through hydrolysis of the N-ribosidic bond using several nucleoside hydrolases. The most abundant nucleoside hydrolase is relatively nonspecific but prefers inosine and uridine as substrates. Here we report the three-dimensional structure of the inosine-uridine nucleoside hydrolase (IU-NH) from C. fasciculata determined by X-ray crystallography at a nominal resolution of 2.5 A. The enzyme has an open (alpha, beta) structure which differs from the classical dinucleotide binding fold. IU-nucleoside hydrolase is composed of a mixed eight-stranded beta sheet surrounded by six alpha helices and a small C-terminal lobe composed of four alpha helices. Two short antiparallel beta strands are involved in intermolecular contacts. The catalytic pocket is located at the C-terminal end of beta strands beta 1 and beta 4. Four aspartate residues are located at the bottom of the cavity in a geometry which suggests interaction with the ribose moiety of the nucleoside. These groups could provide the catalytically important interactions to the ribosyl hydroxyls and the stabilizing anion for the oxycarbonium-like transition state. Histidine 241, located on the side of the active site cavity, is the proposed proton donor which facilitates purine base departure [Gopaul, D. N., Meyer, S. L., Degano, M., Sacchettini, J. C., & Schramm, V. L. (1996) Biochemistry 35, 5963-5970]. The substrate binding site is unlike that from purine nucleoside phosphorylase, phosphoribosyltransferases, or uracil DNA glycosylase and thus represents a novel architecture for general acid-base catalysis. This detailed knowledge of the architecture of the active site, together with the previous transition state analysis [Horenstein, B. A., Parkin, D. W., Estupiñán, B., & Schramm, V. L. (1991) Biochemistry 30, 10788-10795], allows analysis of the interactions leading to catalysis and an explanation for the tight-binding inhibitors of the enzyme [Schramm, V. L., Horenstein, B. A., & Kline, P. C. (1994) J. Biol. Chem. 269, 18259-18262].

Amino Acid Sequence↗
PubMed1996

A polymorphism in the human intestinal fatty acid binding protein alters fatty acid transport across Caco-2 cells.

The human intestinal fatty acid binding protein (IFABP) binds long-chain fatty acids in vitro, but its intracellular function has remained speculative. A polymorphism in the gene that encodes IFABP results in an alanine (Ala54) to threonine (Thr54) substitution at codon 54 that alters the in vitro binding affinity of the protein for long-chain fatty acids. To identify potential functional variability between Ala54 and Thr54 IFABP, we established permanently transfected Caco-2 cell lines that express either Ala54 or Thr54 IFABP. We found that Caco-2 cells expressing Thr54 IFABP transport long-chain fatty acids and secrete triglycerides to a greater degree than Caco-2 cells expressing Ala54 IFABP. These results provide the first demonstration that IFABP participates in the intracellular transport of long-chain fatty acids. In addition, the observed increase in transport of fatty acids across cells expressing Thr54 IFABP suggests a plausible physiologic mechanism for our prior observation that Pima Indians with a Thr54 IFABP genotype have increased post-absorptive lipid oxidation rates and are more insulin-resistant than Pimas with a Ala54 IFABP genotype.

Biological Transport↗
PubMed1996

Molecular mapping of the 8.12 SLE-associated idiotype specificity at the single amino acid level.

The 8.12 idiotype is expressed in elevated titer in the serum of patients with systemic lupus erythematosus and is a marker for a subpopulation of anti-DNA antibodies that possess a V(lambda)II encoded light chain. This study utilized a eukaryotic expression system to identify the structural basis for expression of this idiotype. Reversion of the 8.12+ DSC light chain to the hslv215.23/DPL11 germline gene reveals that the 8.12 idiotype is encoded in the germline. The 8.12+ DSC and the 8.12 AS17 light chains, both belonging to the V(lambda)II family, were subjected to site directed mutagenesis, to localize amino acids important for expression of the 8.12 idiotype. Point mutations were performed in CDR1, CDR2, FR3 and CDR3, in positions where the 8.12+ DSC differs from the 8.12-AS17. Amino acids in CDR1 and the CDR2 proximal region of FR3, but not the J proximal region of CDR3, play a crucial role in 8.12 reactivity. The 3-D structure of Mcg, a human IgG1, with which DSC shares a sequence homology of 92.3% has been examined to visualize the effect of each of the mutations and to identify the surface on DSC that comprises the idiotype.

Amino Acid Sequence↗
PubMed1995

The crystal structure of the orotate phosphoribosyltransferase complexed with orotate and alpha-D-5-phosphoribosyl-1-pyrophosphate.

The three-dimensional structure of Salmonella typhimurium orotate phosphoribosyltransferase (OPRTase) in complex with the ribose 5-phosphate donor alpha-D-5--phosphoribosyl-1-pyrophosphate (PRPP) and the nitrogenous base orotic acid has been solved and refined with X-ray diffraction data extending to 2.3 A resolution to a crystallographic R-factor of 18.7%. The complex was generated by carrying out catalysis in the crystal. Comparison of this structure with the previously reported structure of the orotidine 5'-monophosphate (OMP) complex [Scapin, G., Grubmeyer, C., and Sacchettini, J. C. (1994) Biochemistry 33, 1287-1294] revealed that the enzyme backbone undergoes only small movements. The most significant differences occur near the active site, at Ala71-Gly74, with the largest difference involving the side chains of Lys73, Val127-Ala133, the 5'-phosphate binding loop, and a long, solvent-exposed loop at the dimer interface. The position of the ribose moiety is, on the other hand, very different in the OMP and PRPP.orotate complexes, with its anomeric carbon moving approximately 7 A across the binding cavity. In the PRPP.orotate complex the highly conserved acidic side chain of Asp124 interacts with the ribose of PRPP, whereas there are no interactions of this aspartate with the substrate in the OMP complex.

Binding Sites↗
PubMed1995

Locations and functional roles of conserved lysine residues in Salmonella typhimurium orotate phosphoribosyltransferase.

Salmonella typhimurium orotate phosphoribosyltransferase (OPRTase) catalyzes the formation of orotidine 5'-monophosphate (OMP) from orotate and alpha-D-5-phosphoribosyl-1-pyrophosphate (PRPP). There are five highly conserved lysine residues (Lys-19, -26, -73, -100, and -103) in S. typhimurium OPRTase. Here, we report the results of mutagenesis and substrate analog studies to investigate the functional roles of these lysines. Together with information from X-ray crystallography [Scapin, G., Grubmeyer, C., & Sacchettini, J. C. (1994) Biochemistry 33, 1287-1294; Scapin, G., Ozturk, D. H., Grubmeyer, C., & Sacchettini, J. C. (1995) Biochemistry 34, 10744-10754], sequence comparisons, and chemical modification [Grubmeyer, C., Segura, E., & Dorfman, R. (1993) J. Biol. Chem. 268, 20299-20304], this work permits the assignment of functions of the five conserved lysines. Lys-19 is external to the active site, and its mutation to glutamine had little effect on enzyme activity. Lys-26 forms a hydrogen bond to OMP at the 3'-hydroxyl group, and its mutation produced 3-10-fold decreases in kcat. Lys-73 extends into the active site, and a conformational change allows it to interact with either the 5'-phosphate of OMP or the 2-hydroxyl and alpha-phosphoryl oxygen of PRPP in their respective substrate complexes. Mutation of Lys-73 produced a 50-100-fold decrease in kcat and an 8-12-fold increase in the KM value for PRPP. Mutation of Lys-100 produced a 5-fold decrease in kcat and a 3-fold increase in the KM for PRPP, consistent with its location within the active site, near the pyrophosphate moiety of PRPP.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1995

Structure and function of Salmonella typhimurium orotate phosphoribosyltransferase: protein complementation reveals shared active sites.

A solvent-exposed loop, comprising residues 98-119 of S. typhimurium orotate phosphoribosyltransferase (OPRTase), is at the subunit interface of the dimeric enzyme, and its amino acid side chains potentially contact active sites on either subunit. A portion of the loop (103-107) appears to be mobile on the basis of the X-ray structures of enzyme.OMP [Scapin, G., Grubmeyer, C., & Sacchettini, J. C. (1994) Biochemistry 33, 1287-1294] and enzyme.PRPP.orotate complexes [Scapin, G., Ozturk, D. H., Grubmeyer, C., & Sacchettini, J. C. (1995) Biochemistry 34, 10744-10754]. Lys-103, which is essential for activity [Ozturk, D. H., Dorfman, R. H. Scapin, G., Sacchettini, J. C., & Grubmeyer, C. (1995) Biochemistry 34, 10755-10763], may thus be functional in the active site formed by the adjacent subunit. Asp-125 is an essential residue that is in the middle of the active site. Equimolar mixtures of the nearly inactive K103A and D125N mutant ORPTase subunits produced approximately 21-23% of the enzymatic activity of the wild-type OPRTase. Heterodimer formation in the complemented mixtures was evidenced by various physical methods. Thus, the active site of OPRTase requires Asp-125 from one subunit and Lys-103 from the adjacent subunit. As predicted from the three-dimensional structure, increased activity resulting from complementation was also observed with mixtures of the K103A mutant and the poorly active K73A and K73Q mutants but not with mixtures of D125N and either K73A or K73Q mutants. Neither K103A nor D125N mutants exhibited negative complementation with the wild-type enzyme. A K103A/D125N double mutant enzyme was also constructed and was able to inactivate wild-type enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

Enzymatic characterization of the target for isoniazid in Mycobacterium tuberculosis.

The inhA gene has been recently shown to encode a common protein target for isoniazid and ethionamide action in Mycobacterium tuberculosis. In this paper, we demonstrate that the M. tuberculosis InhA protein catalyzes the NADH-specific reduction of 2-trans-enoyl-ACP, essential for fatty acid elongation. This enzyme preferentially reduces long-chain substrates (12-24 carbons), consistent with its involvement in mycolic acid biosynthesis. Steady-state kinetic studies showed that the two substrates bind to InhA via a sequential kinetic mechanism, with the preferred ordered addition of NADH and the enoyl substrate. The chemical mechanism involves stereospecific hydride transfer of the 4S hydrogen of NADH to the C3 position of the 2-trans-enoyl substrate, followed by protonation at C2 of an enzyme-stabilized enolate intermediate. Kinetic and microcalorimetric analysis demonstrates that the binding of NADH to the S94A mutant InhA, known to confer resistance to both isoniazid and ethionamide, is altered. This difference can account for the isoniazid-resistance phenotype, with the formation of a binary InhA-NADH complex required for drug binding. Isoniazid binding to either the wild-type or S94A mutant InhA could not be detected by titration microcalorimetry, suggesting that this compound is a prodrug, which must be converted to its active form.

Acyl Coenzyme A↗
PubMed1995

X-ray crystal structure of the soybean agglutinin cross-linked with a biantennary analog of the blood group I carbohydrate antigen.

Soybean agglutinin (SBA) (Glycine max), which is a tetrameric GalNAc/Gal-specific lectin, has recently been reported to form unique, highly organized cross-linked complexes with a series of naturally occurring and synthetic multiantennary carbohydrates with terminal GalNAc or Gal residues [Gupta, D., Bhattacharyya, L., Fant, J., Macaluso, F., Sabesan, S., & Brewer, C. F. (1994) Biochemistry 33, 7495-7504]. In order to elucidate the nature of these complexes, the X-ray crystallographic structure of SBA cross-linked with a biantennary analog of the blood group I carbohydrate antigen is reported. The structure reveals that lattice formation is promoted uniquely by the bridging action of the bivalent pentasaccharide (beta-LacNAc)2Gal-beta-R, where R is -O(CH2)5COOCH3 and the beta-LacNAc moieties are linked to the 2 and 6 positions of the core Gal. The structure of SBA complexed with the synthetic biantennary pentasaccharide has thus been determined by molecular replacement techniques and refined at 2.6 A resolution to an R value of 20.1%. The crystals are hexagonal with a P6(4)22 space group, which differs significantly from that of crystals of the free protein. In the structure, each monomeric asymmetric unit contains a Man9 oligomannose-type chain at Asn 75, with only the first two GlcNAc residues visible. The overall tertiary structure of the SBA subunit is similar to that of other legume lectins as well as certain animal lectins. However, the dimer interface in the SBA tetramer is unusual in that only one complete peptide chain is sterically permitted, thus requiring juxtapositioning of one C-terminal fragmented subunit together with an intact subunit. Association between SBA tetramers involves binding of the terminal Gal residues of the pentasaccharide at identical sites in each monomer, with the sugar cross-linking to a symmetry-related neighbor molecule. The cross-linking pentasaccharide is in a conformation that possesses a pseudo-2-fold axis of symmetry which lies on a crystallographic 2-fold axis of symmetry of the lattice. Hence, the symmetry properties of the bivalent oligosaccharide as well as the lectin are structural determinants of the lattice. The results are discussed in terms of multidimensional carbohydrate-lectin cross-linked complexes, as well as the signal transduction properties of multivalent lectins.

Binding Sites↗