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Biomedical subjects

B L Stoddard

Publications and source records attributed to B L Stoddard.

At least 37 records · Page 2Linked to original sources
PubMed1998

The allosteric regulation of pyruvate kinase by fructose-1,6-bisphosphate.

BACKGROUND: Yeast pyruvate kinase (PK) catalyzes the final step in glycolysis. The enzyme therefore represents an important control point and is allosterically activated by fructose-1,6-bisphosphate (FBP). In mammals the enzyme is found as four different isozymes with different regulatory properties: two of these isozymes are produced by alternate splicing. The allosteric regulation of PK is directly related to proliferation of certain cell types, as demonstrated by the expression of an allosterically regulated isozyme in tumor cells. A model for the allosteric transition from the inactive (T) state to the active (R) state has been proposed previously, but until now the FBP-binding site had not been identified. RESULTS: We report here the structures of PK from yeast complexed with a substrate analog and catalytic metal ions in the presence and absence of bound FBP. The allosteric site is located 40 A from the active site and is entirely located in the enzyme regulatory (C) domain. A phosphate-binding site for the allosteric activator is created by residues encoded by a region of the gene corresponding to the alternately spliced exon of mammalian isozymes. FBP activation appears to induce several conformational changes among active-site sidechains through a mechanism that is most likely to involve significant domain motions, as previously hypothesized. CONCLUSIONS: The structure and location of the allosteric activator site agrees with the pattern of alternate genetic splicing of the PK gene in multicellular eukaryotes that distinguishes between a non-regulated isozyme and the regulated fetal isozymes. The conformational differences observed between the active sites of inactive and fully active PK enzymes is in agreement with the recently determined thermodynamic mechanism of allosteric activation through a 'metal relay' that increases the affinity of the enzyme for its natural phosphoenolpyruvate substrate.

Allosteric Regulation↗
PubMed1998

New results using Laue diffraction and time-resolved crystallography.

Several time-resolved crystallographic structures were determined over the past year, using a variety of trapping protocols and several data collection methods. A significant theme of recent time-resolved work is the importance of parallel comparative studies on the same protein, using different experimental protocols, in order to fully characterize the structural variation of the intermediates formed in the reaction pathway.

Bacterial Proteins↗
PubMed1998

DNA recognition and cleavage by the LAGLIDADG homing endonuclease I-CreI.

The structure of the LAGLIDADG intron-encoded homing endonuclease I-CreI bound to homing site DNA has been determined. The interface is formed by an extended, concave beta sheet from each enzyme monomer that contacts each DNA half-site, resulting in direct side-chain contacts to 18 of the 24 base pairs across the full-length homing site. The structure indicates that I-CreI is optimized to its role in genetic transposition by exhibiting long site-recognition while being able to cleave many closely related target sequences. DNA cleavage is mediated by a compact pair of active sites in the I-CreI homodimer, each of which contains a separate bound divalent cation.

Binding Sites↗
PubMed1998

Millisecond Laue structures of an enzyme-product complex using photocaged substrate analogs.

The structure of a rate-limited product complex formed during a single initial round of turnover by isocitrate dehydrogenase has been determined. Photolytic liberation of either caged substrate or caged cofactor and Laue X-ray data collection were used to visualize the complex, which has a minimum half-life of approximately 10 milliseconds. The experiment was conducted with three different photoreactive compounds, each possessing a unique mechanism leading to the formation of the enzyme-substrate (ES) complex. Photoreaction efficiency and subsequent substrate affinities and binding rates in the crystal are critical parameters for these experiments. The structure suggests that CO2 dissociation is a rapid event that may help drive product formation, and that small conformational changes may contribute to slow product release.

Catalysis↗
PubMed1997

Caged NADP and NAD. Synthesis and characterization of functionally distinct caged compounds.

Two caged NADP compounds have been synthesized and characterized for use in the crystallographic study of isocitrate dehydrogenase (IDH), as well as for general use in cell biology, metabolism, and enzymology. One caged NADP compound has been designed to be "catalytically caged" so that it can bind to IDH prior to photolysis but is not catalytically active. A second NADP compound is "affinity caged" so that addition of the caging group inhibits binding of the compound to IDH prior to photolysis. The catalytically caged compound was synthesized in a two-step process, starting with the NADase-catalyzed exchange of a synthetic nicotinamide derivative onto NADP. X-ray structures of the NADP compounds with IDH show the catalytically caged NADP bound to the enzyme with its nicotinamide group improperly positioned to allow turnover, while the affinity caged NADP does not bind to the enzyme at concentrations up to 50 mM. Two analogous caged NAD compounds have also been synthesized. The NADP and NAD compounds were characterized in terms of kinetics, quantum yield, and product formation. The affinity caged NADP compound P2'-[1-(4,5-dimethoxy-2-nitrophenyl)ethyl] NADP (VIII) is photolyzed at a rate of 1.8 x 10(4) s-1 with a quantum yield of 0.19 at pH 7; the NAD analog P-[1-(4,5-dimethoxy-2-nitrophenyl)ethyl] NAD (IX) is photolyzed at at a rate of 1.7 x 10(4) s-1 with a quantum yield of 0.17.

Binding Sites↗
PubMed1997

Orbital steering in the catalytic power of enzymes: small structural changes with large catalytic consequences.

Small structural perturbations in the enzyme isocitrate dehydrogenase (IDH) were made in order to evaluate the contribution of precise substrate alignment to the catalytic power of an enzyme. The reaction trajectory of IDH was modified (i) after the adenine moiety of nicotinamide adenine dinucleotide phosphate was changed to hypoxanthine (the 6-amino was changed to 6-hydroxyl), and (ii) by replacing Mg2+, which has six coordinating ligands, with Ca2+, which has eight coordinating ligands. Both changes make large (10(-3) to 10(-5)) changes in the reaction velocity but only small changes in the orientation of the substrates (both distance and angle) as revealed by cryocrystallographic trapping of active IDH complexes. The results provide evidence that orbital overlap produced by optimal orientation of reacting orbitals plays a major quantitative role in the catalytic power of enzymes.

Cadmium↗
PubMed1997

Purification, crystallization, and preliminary X-ray studies of 10-formyltetrahydrofolate synthetase from Clostridia acidici-urici.

The monofunctional enzyme 10-formyltetrahydrofolate synthetase (THFS), which is responsible for the recruitment of single carbon units from the formate pool into a variety of folate-dependent biosynthetic pathways, has been subcloned, purified, and crystallized. The crystals belong to space group P2(1), with unit cell dimensions a = 102.4 A, b = 116.5 A, c = 115.8 A, and beta = 103.5. The crystal unit cell and diffraction is consistent with an asymmetric unit consisting of the enzyme tetramer, and a specific volume of the unit cell of 2.7 A3/ Da. The crystals diffract to at least 2.3 A resolution after flash-cooling, when using a rotating anode x-ray source and an RAXIS image plate detector.

Bacterial Proteins↗
PubMed1997

Purification, crystallization, and preliminary x-ray studies of a bifunctional 5,10-methenyl/methylene-tetrahydrofolate cyclohydrolase/dehydrogenase from Escherichia coli.

A bifunctional enzyme that catalyzes the conversion of formyltetrahydrofolate to methylene-tetrahydrofolate (5,10-methenyltetrahydrofolate cyclohydrolase and 5,10-methylene tetrahydrofolate dehydrogenase), has been subcloned from a cDNA library, purified to homogeneity, and crystallized. The crystals belong to space group I222, with unit cell dimensions of a = 64.5 A, b = 84.9 A, c = 146.1 A. The crystal unit cell and diffraction is consistent with an asymmetric unit consisting of the enzyme monomer, and a specific volume of the unit cell of 3.2 A3/Da. The crystals diffract to at least 2.8 A resolution after flash-cooling, when using a rotating anode x-ray source and an RAXIS image plate detector. A 2.56 A resolution native data set has been collected at beamline X12-C at the NSLS.

Aminohydrolases↗
PubMed1997

Crystallization and preliminary X-ray studies of I-CreI: a group I intron-encoded endonuclease from C. reinhardtii.

Group I intron endonuclease I-CreI is encoded by an open reading frame contained within a self-splicing intron in the Chlamydomonas reinhardtii chloroplast 23S rRNA gene. I-CreI initiates the lateral transfer or homing of this intron by specifically recognizing and cleaving a pseudopalindromic 19-24 bp homing site in chloroplast 23S rRNA genes that lack the intron. The gene encoding this enzyme has been subcloned, and the protein product has been purified and crystallized. The crystals belong to space group P321, with unit cell dimensions a = b = 78.2 A, c = 67.4 A. The crystal unit cell is consistent with an asymmetric unit consisting of the enzyme monomer. The specific volume of this unit cell is 3.3 A3/Da. The crystals diffract to at least 3.0 A resolution after flash-cooling, when using a rotating anode x-ray source and an RAXIS image plate detector.

Animals↗
PubMed1997

Crystallization and preliminary X-ray studies of I-PpoI: a nuclear, intron-encoded homing endonuclease from Physarum polycephalum.

The homing endonuclease I-PpoI is encoded by an optional third intron, Pp LSU 3, found in nuclear, extrachromosomal copies of the Physarum polycephalum 26S rRNA gene. This endonuclease promotes the lateral transfer or "homing" of its encoding intron by recognizing and cleaving a partially symmetric, 15 bp homing site in 26S rDNA alleles that lack the Pp LSU 3 intron. The open reading frame encoding I-PpoI has been subcloned, and the endonuclease has been overproduced in E. coli. Purified recombinant I-PpoI has been co-crystallized with a 21 bp homing site DNA duplex. The crystals belong to space group P3(1)21, with unit cell dimensions a = b = 114 A, c = 89 A. The results of initial X-ray diffraction experiments indicate that the asymmetric unit contains an enzyme homodimer and one duplex DNA molecule, and that the unit cell has a specific volume of 3.4 A3/dalton. These experiments also provide strong evidence that I-PpoI contains several bound zinc ions as part of its structure.

Animals↗
PubMed1997

The structure of I-Crel, a group I intron-encoded homing endonuclease.

The structure of I-Crel provides the first view of a protein encoded by a gene within an intron. This endonuclease recognizes a long DNA site approximately 20 base pairs in length and facilitates the lateral transfer of that intron. The protein exhibits a DNA-binding surface consisting of four antiparallel beta-strands that form a 20 A wide groove which is over 70 A long. The architecture of this fold is different from that of the TATA binding protein, TBP, which also contains an antiparallel beta-saddle. The conserved LAGLIDADG motif, which is found in many mobile intron endonucleases, maturases and inteins, forms a novel helical interface and contributes essential residues to the active site.

Amino Acid Sequence↗
PubMed1996

Capturing the structure of a catalytic RNA intermediate: the hammerhead ribozyme.

The crystal structure of an unmodified hammerhead RNA in the absence of divalent metal ions has been solved, and it was shown that this ribozyme can cleave itself in the crystal when divalent metal ions are added. This biologically active RNA fold is the same as that found previously for two modified hammerhead ribozymes. Addition of divalent cations at low pH makes it possible to capture the uncleaved RNA in metal-bound form. A conformational intermediate, having an additional Mg(II) bound to the cleavage-site phosphate, was captured by freeze-trapping the RNA at an active pH prior to cleavage. The most significant conformational changes were limited to the active site of the ribozyme, and the changed conformation requires only small additional movements to reach a proposed transition-state.

Base Sequence↗
PubMed1996

Intermediate trapping and laue X-ray diffraction: potential for enzyme mechanism, dynamics, and inhibitor screening.

Among the many macromolecules of biomedical interest, enzyme catalysts remain important targets for the development of inhibitors and drugs, due to our ability to directly measure inhibition constants in vitro. Crystallographic structures of enzymes allow drug screening to be carried out by computational analysis of small molecules for structural and chemical complementarity to the active site. Such methods generally rely on static target structures. The development of time-resolved crystallographic methods, including trapping of reaction intermediates and Laue diffraction, allow the comparative study of enzyme conformations at different intermediate states in the catalytic cycle, providing an experimental avenue for visualizing and exploiting discrete structural states. Such methods have clear implications for mechanistic studies and possibly for computational drug design.

Chymotrypsin↗
PubMed1996

Calcineurin-immunosuppressor complexes.

Crystal structures of the Ser/Thr phosphatase calcineurin (protein phosphatase 2B) have recently been solved by X-ray crystallography, both in the free-protein state, and complexed with the immunophilin/immunosuppressant FKBP12/FK506. Core elements of the calcineurin phosphatase have been found to be similar to the corresponding elements of Ser/Thr phosphatase 1 and purple acid phosphatase. The structures provide a basis for understanding calcineurin inhibition by a ternary complex of immunophilin and immunosuppressant proteins.

Binding Sites↗
PubMed1996

Combining Laue diffraction and molecular dynamics to study enzyme intermediates.

Two separate techniques, Laue diffraction and computational molecular dynamics (MD) simulations, have been independently developed to allow the visualization and assessment of transient structural states. Recent studies on isocitrate dehydrogenase show that computational MD simulations of an enzymatic Michaelis complex are consistent with difference Fourier electron density maps of the same structure from a Laue experiment. The use of independent MD studies during crystallographic refinement has allowed us to assign with confidence a number of additional contacts and features important for hydride transfer. We find that unrestrained independent MD simulations provides a very useful method of cross-validation for highly mobile atoms in regions of experimental density that are poorly defined. Likewise, information from Laue difference maps provides information about substrate conformation and interactions that greatly facilitate MD simulations.

Computer Simulation↗