Dihydrofolate reductase: x-ray structure of the binary complex with methotrexate.

A central eight-stranded beta-pleated sheet is the main feature of the polypeptide backbone folding in dihydrofolate reductase. The innermost four strands and two bridging helices are geometrically similar to but are connected in a different way from those in the dinucleotide binding domains found in nicotinamide-adenine dinucleotide-linked dehydrogenases. Methotrexate is bound in a 15-angstrom-deep cavity with the pteridine ring buried in a primarily hydrophobic pocket, although a strong interaction occurs between the side chain of aspartic acid 27 and N(1), N(8), and the 2-amino group of methotrexate.

A portable magneto-optical trap with prospects for atom interferometry in civil engineering.

The high precision and scalable technology offered by atom interferometry has the opportunity to profoundly affect gravity surveys, enabling the detection of features of either smaller size or greater depth. While such systems are already starting to enter into the commercial market, significant reductions are required in order to reach the size, weight and power of conventional devices. In this article, the potential for atom interferometry based gravimetry is assessed, suggesting that the key opportunity resides within the development of gravity gradiometry sensors to enable drastic improvements in measurement time. To push forward in realizing more compact systems, techniques have been pursued to realize a highly portable magneto-optical trap system, which represents the core package of an atom interferometry system. This can create clouds of 107 atoms within a system package of 20 l and 10 kg, consuming 80 W of power.This article is part of the themed issue 'Quantum technology for the 21st century'.

Stereochemical mechanism of action for thymidylate synthase based on the X-ray structure of the covalent inhibitory ternary complex with 5-fluoro-2'-deoxyuridylate and 5,10-methylenetetrahydrofolate.

The structure of the Escherichia coli thymidylate synthase (TS) covalent inhibitory ternary complex consisting of enzyme, 5-fluoro-2'-deoxyuridylate (FdUMP) and 5,10-methylene tetrahydrofolate (CH2-H4PteGlu) has been determined at 2.5 A resolution using difference Fourier methods. This complex is believed to be a stable structural analog of a true catalytic intermediate. Knowledge of its three-dimensional structure and that for the apo enzyme, also reported here, suggests for the first time how TS may activate dUMP and CH2-H4PteGlu leading to formation of the intermediate and offers additional support for the hypothesis that the substrate and cofactor are linked by a methylene bridge between C-5 of the substrate nucleotide and N-5 of the cofactor. By correlating these structural results with the known stereospecificity of the TS-catalyzed reaction it can be inferred that the catalytic intermediate, once formed, must undergo a conformational isomerization before eliminating across the bond linking C-5 of dUMP to C-11 of the cofactor. The elimination itself may be catalyzed by proton transfer to the cofactor's 5 nitrogen from invariant Asp169 buried deep in the TS active site. The juxtaposition of Asp169 and bound tetrahydrofolate in TS is remarkably reminiscent of binding geometry found in dihydrofolate reductase where a similarly conserved carboxyl group serves as a general acid for protonating the corresponding pyrazine ring nitrogen of dihydrofolate.

Molecular recognition of the inhibitor AG-1343 by HIV-1 protease: conformationally flexible docking by evolutionary programming.

BACKGROUND: An important prerequisite for computational structure-based drug design is prediction of the structures of ligand-protein complexes that have not yet been experimentally determined by X-ray crystallography or NMR. For this task, docking of rigid ligands is inadequate because it assumes knowledge of the conformation of the bound ligand. Docking of flexible ligands would be desirable, but requires one to search an enormous conformational space. We set out to develop a strategy for flexible docking by combining a simple model of ligand-protein interactions for molecular recognition with an evolutionary programming search technique. RESULTS: We have developed an intermolecular energy function that incorporates steric and hydrogen-bonding terms. The parameters in this function were obtained by docking in three different protein systems. The effectiveness of this method was demonstrated by conformationally flexible docking of the inhibitor AG-1343, a potential new drug against AIDS, into HIV-1 protease. For this molecule, which has nine rotatable bonds, the crystal structure was reproduced within 1.5 A root-mean-square deviation 34 times in 100 simulations, each requiring eight minutes on a Silicon Graphics R4400 workstation. The energy function correctly evaluates the crystal structure as the global energy minimum. CONCLUSIONS: We believe that a solution of the docking problem may be achieved by matching a simple model of molecular recognition with an efficient search procedure. The necessary ingredients of a molecular recognition model include only steric and hydrogen-bond interaction terms. Although these terms are not necessarily sufficient to predict binding affinity, they describe ligand-protein interactions faithfully enough to enable a docking program to predict the structure of the bound ligand. This docking strategy thus provides an important tool for the interdisciplinary field of rational drug design.

Quantifying the quantum gate fidelity of single-atom spin qubits in silicon by randomized benchmarking.

Building upon the demonstration of coherent control and single-shot readout of the electron and nuclear spins of individual (31)P atoms in silicon, we present here a systematic experimental estimate of quantum gate fidelities using randomized benchmarking of 1-qubit gates in the Clifford group. We apply this analysis to the electron and the ionized (31)P nucleus of a single P donor in isotopically purified (28)Si. We find average gate fidelities of 99.95% for the electron and 99.99% for the nuclear spin. These values are above certain error correction thresholds and demonstrate the potential of donor-based quantum computing in silicon. By studying the influence of the shape and power of the control pulses, we find evidence that the present limitation to the gate fidelity is mostly related to the external hardware and not the intrinsic behaviour of the qubit.

De novo design of enzyme inhibitors by Monte Carlo ligand generation.

A new computational method for the in situ generation of small molecules within the binding site of a protein is described. The method has been evaluated using two well-studied systems, dihydrofolate reductase and thymidylate synthase. The method has also been used to guide improvements to inhibitors of HIV-1 protease. One such improvement resulted in a compound selected for preclinical studies as an antiviral agent against AIDS.

The influence of carbon and nitrogen nutrition on fusarin C biosynthesis by Fusarium moniliforme.

The influence of various carbon and nitrogen sources on fusarin C synthesis was examined in submerged cultures of Fusarium moniliforme NRRL 13616. Using a zinc-deficient, synthetic medium, highest levels of fusarin C were produced by cultures grown with urea or ammonium sulfate as the nitrogen source and fructose, sucrose, or glucose as the carbon source. In media supplemented with various concentrations of glucose and ammonium sulfate, glucose concentrations which provided excess carbohydrate significantly increased fusarin C synthesis, regardless of the ammonium sulfate concentration.

Use of an oral assessment tool to improve practice.

An oral assessment tool was developed alongside care plans in a neurosciences unit, including the rationale for interventions, to provide nurses with an evidence-based means of providing mouth care. Awareness and standards of mouth care improved as a result of introducing the tool and care plans.

Purification and characterization of the extracellular beta-glucosidase produced by Candida wickerhamii.

Candida wickerhamii NRRL Y-2563 produced a cell-bound beta-glucosidase when grown in complex media containing 50 g of cellobiose per liter. The majority of the enzyme was located on the cell surface and was released into the supernatant upon treatment of intact cells with Zymolyase 60,000. Only about 10% of the total activity was associated with the cytoplasm. The enzyme was purified to homogeneity, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme had an apparent native molecular mass of about 198,000 Da and appeared to be composed of two subunits with approximate molecular masses of 94,000 Da. The beta-glucosidase contained approximately 30.5% (w/w) carbohydrate. Mannose was the only detected neutral carbohydrate associated with the purified enzyme. The enzyme demonstrated optimal activity at a pH of 4.0 to 5.0. The Km of the purified beta-glucosidase was 6.74 X 10(-2) M for cellobiose and 4.17 X 10(-3) M for p-nitrophenyl-beta-D-glucopyranoside. Glucose did not appear to inhibit the enzyme.

Fermentation and aerobic metabolism of cellodextrins by yeasts.

The fermentation and aerobic metabolism of cellodextrins by 14 yeast species or strains was monitored. When grown aerobically, Candida wickerhamii, C. guilliermondii, and C. molischiana metabolized cellodextrins of degree of polymerization 3 to 6. C. wickerhamii and C. molischiana also fermented these substrates, while C. guilliermondii fermented only cellodextrins of degree of polymerization less than or equal to 3. Debaryomyces polymorphus, Pichia guilliermondii, Clavispora lusitaniae, and one of two strains of Kluyveromyces lactis metabolized glucose, cellobiose, and cellotriose when grown aerobically. These yeasts also fermented these substrates, except for K. lactis, which fermented only glucose and cellobiose. The remaining species/strains tested, K. lactis, Brettano-myces claussenii, B. anomalus, K. dobzhanskii, Rhodotorula minuta, and Dekkera intermedia, both fermented and aerobically metabolized glucose and cellobiose. Crude enzyme preparations from all 14 yeast species or strains were tested for ability to hydrolyze cellotriose and cellotretose. Most of the yeasts produced an enzyme(s) capable of hydrolyzing cellotriose. However, with two exceptions, R. minuta and P. guilliermondii, only the yeasts that metabolized cellodextrins of degree of polymerization greater than 3 produced an enzyme(s) that hydrolyzed cellotretose.

Purification and characterization of the extracellular alpha-amylase from Streptococcus bovis JB1.

The extracellular alpha-amylase (1,4-alpha-D-glucanglucanohydrolase; EC 3.2.1.1) from maltose-grown Streptococcus bovis JB1 was purified to apparent homogeneity by ion-exchange chromatography (Mono Q). The enzyme had an isoelectric point of 4.50 and an apparent molecular mass of 77,000 Da, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was rich in acidic and hydrophobic amino acids. The 15-amino-acid NH2-terminal sequence was 40% homologous with the Bacillus subtilis saccharifying alpha-amylase and 27% homologous with the Clostridium acetobutylicum alpha-amylase. alpha-Amylase activity on soluble starch was optimal at pH 5.0 to 6.0. The enzyme was relatively stable between pH 5.5 and 8.5 and at temperatures below 50 degrees C. When soluble potato starch was used as the substrate, the enzyme had a Km of 0.88 mg.ml-1 and a kcat of 2,510 mumol of reducing sugar.min-1.mg of protein-1. The enzyme exhibited neither pullulanase nor dextranase activity and was 40 to 70% as active on amylopectin as on amylose. The major end products of amylose hydrolysis were maltose, maltotriose, and maltotetraose.

Kinetic characterization of a beta-glucosidase from a yeast, Candida wickerhamii.

The extracytoplasmic, cell-bound beta-1,4-glucosidase of Candida wickerhamii was characterized kinetically. The enzyme was found to produce glucose from cellobiose and cellodextrins (degree of polymerization from three to six) by catalyzing the removal of the terminal glucose moiety from the nonreducing end of these beta-glucans. The Km values for the series, cellobiose through cellohexaose, were 210.7, 106.6, 106.3, 105.9, and 79.8 mM, respectively, whereas the kcat values were 14.79, 13.24, 13.78, 15.13, and 7.66 mumol of glucose.min-1.mg-1 of protein, respectively. A computer program was developed to estimate the integrated rate equation. When the above kinetic constants were used in the computer model, the predicted rates of glucose formation agreed well with the experimental data. Saccharomyces cerevisiae, which is unable to ferment cellobiose or cellodextrins, ferments glucose about twice as fast as C. wickerhamii. If S. cerevisiae is cultured on cellobiose or cellodextrins and the purified C. wickerhamii beta-glucosidase is added to the S. cerevisiae culture at levels that mimic the production of beta-glucosidase by a C. wickerhamii culture with time, the two cultures produce ethanol at equivalent rates. This suggests that the rate-limiting step in the fermentation of cellobiose/cellodextrins by C. wickerhamii is the production of beta-glucosidase.

Regulation of beta-1, 4-Glucosidase Expression by Candida wickerhamii.

Candida wickerhamii NRRL Y-2563 expressed beta-glucosidase activity (3 to 8 U/ml) constitutively when grown aerobically in complex medium containing either glycerol, succinate, xylose, galactose, or cellobiose as the carbon source. The addition of a high concentration of glucose (>75 g/liter) repressed beta-glucosidase expression (<0.3 U/ml); however, this yeast did produce beta-glucosidase when the initial glucose concentration was

Cloning and characterization of a gene encoding a cell-bound, extracellular beta-glucosidase in the yeast Candida wickerhamii.

The ability of yeasts to ferment cellodextrins is rare. Candida wickerhamii is able to use these sugars for alcohol production because of a cell-bound, extracellular, beta-glucosidase that is unusual by not being inhibited by glucose. A cDNA expression library in lambda phage was prepared with mRNA isolated from cellobiose-grown C. wickerhamii. Immunological screening of the library with polyclonal antibodies against purified C. wickerhamii cell-bound, extracellular beta-glucosidase yielded 12 positive clones. Restriction endonuclease analysis and sequence data revealed that the clones could be divided into two groups, bglA and bglB, which were shown to be genetically distinct by Southern hybridization analyses. Efforts were directed at the study of bglB since it appeared to code for the cell-bound beta-glucosidase. Sequence data from both cDNA and genomic clones showed the absence of introns in bglB. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting of cell lysates from Escherichia coli bglB clones confirmed the presence of an expressed protein with an apparent molecular mass of 72 kDa, which is consistent with that expected for an unglycosylated form of the enzyme. Amino acid comparisons of BglB with other beta-glucosidase sequences suggest that it is a member of family 1 glycosyl hydrolases but is unusual in that it contains an additional 100 to 130 amino acids at the N terminus. This sequence did not have homologies to other known protein sequences and may impart unique properties to this beta-glucosidase.