X-ray crystallographic study of xylopentaose binding to Pseudomonas fluorescens xylanase A.

The structure of the complex between a catalytically compromised family 10 xylanase and a xylopentaose substrate has been determined by X-ray crystallography and refined to 3.2 A resolution. The substrate binds at the C-terminal end of the eightfold betaalpha-barrel of Pseudomonas fluorescens subsp. cellulosa xylanase A and occupies substrate binding subsites -1 to +4. Crystal contacts are shown to prevent the expected mode of binding from subsite -2 to +3, because of steric hindrance to subsite -2. The loss of accessible surface at individual subsites on binding of xylopentaose parallels well previously reported experimental measurements of individual subsites binding energies, decreasing going from subsite +2 to +4. Nine conserved residues contribute to subsite -1, including three tryptophan residues forming an aromatic cage around the xylosyl residue at this subsite. One of these, Trp 313, is the single residue contributing most lost accessible surface to subsite -1, and goes from a highly mobile to a well-defined conformation on binding of the substrate. A comparison of xylanase A with C. fimi CEX around the +1 subsite suggests that a flatter and less polar surface is responsible for the better catalytic properties of CEX on aryl substrates. The view of catalysis that emerges from combining this with previously published work is the following: (1) xylan is recognized and bound by the xylanase as a left-handed threefold helix; (2) the xylosyl residue at subsite -1 is distorted and pulled down toward the catalytic residues, and the glycosidic bond is strained and broken to form the enzyme-substrate covalent intermediate; (3) the intermediate is attacked by an activated water molecule, following the classic retaining glycosyl hydrolase mechanism.

Measurement of plasma volume using 99Tc(m)-labelled DMP-HSA.

Red cell volume (RCV) and plasma volume (PV) measurements are performed routinely in nuclear medicine departments to diagnose a number of haematological disorders. Currently, 125I-HSA is used as a plasma tracer and 99Tcm-labelled red cells to determine red cell volume. 125I-HSA is not always readily available, leading to inconvenience for patients and medical practitioners. Due to the availability of 99Tcm in nuclear medicine departments, the use of albumin labelled with 99Tcm was investigated. A new 99Tcm-human serum albumin labelling kit (99Tcm-DMP-HSA) was developed by Verbeke and supplied for use in this study. The main aim of the study was to investigate the use of 99Tcm-DMP-HSA for PV determination. Secondly, the feasibility to determine red cell and plasma volume simultaneously using 99Tcm as radionuclide in both instances was investigated. Fourteen healthy volunteers were enrolled in the dual-phase study. During the first study, 99Tcm-DMP-HSA was used as tracer to calculate PV (PV1a) after intravenous administration. Subsequently, 99Tcm-labelled red cells were administered and the PV (PV1b) and RCV (RCV1) were calculated. The second study was repeated within 2 weeks using the conventional method. 125I-HSA and 99Tcm-labelled red cells were administered simultaneously. The PV (PV2) and RCV (RCV2) were calculated. We found that the redistribution of 99Tcm-DMP-HSA is faster than that of 125I-HSA; therefore, the plasma counts obtained at different times were back-extrapolated to time zero for plasma volume calculations. The mean values for the different calculated PVs were 2964+/-470 ml for PV1a, 3006+/-623 ml for PV1b and 3001+/-530 ml for PV2, the reference PV. The confidence intervals indicate no significant differences between plasma volumes PV1a and PV2 and plasma volumes PV1a and PV1b. The mean calculated RCV1 was 2130+/-322 ml; that of RCV2 was 2128+/-353 ml. The difference between RCV1 and RCV2 was not significant. Our results indicate that 99Tcm-DMP-HSA could be used for plasma volume calculation. Red cell and plasma volumes can be calculated simultaneously using 99Tcm as radionuclide in both cases.

A single domain thermophilic xylanase can bind insoluble xylan: evidence for surface aromatic clusters.

A clone expressing xylanase activity in Escherichia coli has been selected from a genomic plasmid library of the thermophilic Bacillus strain D3. Subcloning from the 9-kb insert located the xylanase activity to a 2.7-kb HindII/BamHI fragment. The DNA sequence of this clone revealed an ORF of 367 codons encoding a single domain type-F or family 10 enzyme, which was designated as XynA. Purification of the enzyme following over-expression in E. coli produced an enzyme of 42 kDa with a temperature optimum of 75 degrees C which can efficiently bind and hydrolyse insoluble xylan. The pH optimum of the enzyme is 6.5, but it is active over a broad pH range. A homology model of the xylanase has been constructed which reveals a series of surface aromatic residues which form hydrophobic clusters. This unusual structural feature is strikingly similar to the situation observed in the structure determined for the type-G xylanase from the Bacillus D3 strain and may constitute a common evolutionary mechanism imposed on different structural frameworks by which these xylanases may bind potential substrates and exhibit thermostability.

Crystallization and preliminary X-ray analysis of arabinanase A from Pseudomonas fluorescens subspecies cellulosa.

Crystals of 1,5-alpha-arabinanase A from Pseudomonas fluorescens subspecies cellulosa have been obtained by vapour diffusion. The crystals belong to the space group P6122 with unit-cell parameters a = b = 91.6, c = 179.4 A with one molecule in the asymmetric unit. The native crystals and, to a much greater extent, heavy-atom soaked crystals are sensitive to radiation which necessitates cryocooling. Suitable cryocooling conditions have been established, though a shrinkage of the unit cell is observed, with a = b = 88.8 and c = 176.9 A.

Crystallization and preliminary X-ray diffraction studies of a family 26 endo-beta-1,4 mannanase (ManA) from Pseudomonas fluorescens subspecies cellulosa.

Crystals of an endo-beta-1,4-mannanase (1,4-beta-D-mannohydrolase, E. C. 3.2.1.78) from Pseudomonas fluorescens sub species cellulosa have been grown by the hanging-drop technique at 291 K over a period of one to two weeks to maximal dimensions of 0.17 x 0.17 x 0.25 mm. These crystals belong to the space group R32 (or R3) with cell dimensions of a = b = 155.4 and c = 250.8 A (hexagonal setting) and contain three (six) molecules in the asymmetric unit. The crystals diffract to at least 3.2 A using a laboratory source and are suitable for structure determination.

Structural basis of the properties of an industrially relevant thermophilic xylanase.

A thermophilic xylanase from Bacillus strain D3 suitable for use as a bleach booster in the paper pulping industry has been identified and characterized. The enzyme is suited to the high temperature and alkaline conditions needed for using xylanases in the pulp industry. The xylanase is stable at 60 degrees C and relatively stable at high temperatures, with a temperature optimum of 75 degrees C. The pH optimum is 6, but the enzyme is active over a broad pH range. The xylanase has been cloned and sequenced, and the crystal structure has been determined. The structure of Bacillus D3 xylanase reveals an unusual feature of surface aromatic residues, which form clusters or "sticky patches" between pairs of molecules. These "sticky patches" on the surface of the enzyme are responsible for the tendency of the protein to aggregate at high concentrations in the absence of reagents such as ethylene glycol. The formation of dimers and higher order polymers via these hydrophobic contacts may also contribute to the thermostability of this xylanase.

Crystallization and preliminary X-ray analysis of arabinofuranosidase C from Aspergillus niger strain 3M43.

Crystals of arabinofuranosidase C purified from Aspergillus niger strain 3M43 have been obtained by vapour diffusion. The crystal belongs to the space group P2(1) with cell parameters a = 44.28, b = 71.99, c = 45.27 A and beta = 105.98 degrees with one molecule in the asymmetric unit. The X-ray diffraction pattern of these crystals extends to at least 2.20 A resolution with the use of synchrotron radiation. These crystals are stable on exposure to radiation and are suitable for structure determination.

Visible intracavity laser spectroscopy with a step-scan Fourier-transform interferometer.

A Fourier-transform spectrometer has been used in a step-scan mode to make time-resolved measurements of the evolving laser pulse in intracavity laser spectroscopy (ILS) experiments. Spectra of broadband dye laser pulses at approximately 615 nm were recorded at relatively high spectral (0.5-cm(-1)) and temporal (as high as 5-mus) resolution. In the absence of an absorber, the height of the pulse is shown to be proportional to t(g)(0.57) (where t(g) is the generation time) for generation times as high as 500 mus. The system was constructed for feasibility studies of future use at infrared and near-infrared wavelengths where conventional ILS that uses diode arrays would be either expensive or simply not possible. The CH(4) overtone transition at 619.68 nm was used to test the linearity and sensitivity of the system. Comparable performance to conventional ILS systems was demonstrated, as were the advantages of the present system for studies of laser and absorption dynamics.

Refined crystal structure of the catalytic domain of xylanase A from Pseudomonas fluorescens at 1.8 A resolution.

The three-dimensional structure of native xylanase A from Pseudomonas flouorescens subspecies cellulosa has been refined at 1.8 A resolution. The space group is P2(1)2(1)2(1) with four molecules in the asymmetric unit. The final model has an R factor of 0.166 for 103 749 reflections with the four molecules refined independently. The tertiary structure consists of an eightfold beta/alpha-barrel, the so-called TIM-barrel fold. The active site is in an open cleft at the carboxy-terminal end of the beta/alpha-barrel, and the active-site residues are a pair of glutamates, Glu127 on strand 4 and Glu246 on strand 7. Both these catalytic glutamate residues are found on beta-bulges. An atypically long loop after strand 7 is stabilized by calcium. Unusual features include a non-proline cis-peptide residue Ala80 which is found on a beta-bulge at the end of beta-strand 3. The three beta-bulge type distortions occurring on beta-strands 3, 4 and 7 are functionally significant as they serve to orient important active-site residues. The active-site residues are further held in place by an extensive hydrogen-bonding network of active-site residues in the catalytic site of xylanase A. A chain of well ordered water molecules occupies the substrate-binding cleft, some or all of which are expelled on binding of the substrate.

Four-laser airborne infrared spectrometer for atmospheric trace gas measurements.

We describe the four-laser airborne infrared (FLAIR) instrument, a tunable diode laser absorption spectrometer designed for simultaneous high-sensitivity in situ measurements of four atmospheric trace gases in the troposphere. The FLAIR spectrometer was employed during the large-scale airborne research campaign on tropospheric ozone (TROPOZ II) in 1991 and was used to measure CO, H(2) O(2), HCHO, and NO(2) in the free troposphere where detection limits below 100 parts in 10(12) by volume were achieved.

Fast ab initio calculation of solvent envelopes for protein structures.

A fast and simple method has been developed for the ab initio calculation of low-resolution solvent envelopes for macromolecular structures. In essence, a sphere of point scatterers is moved through the asymmetric unit cell in a part random, part systematic search for the configuration which corresponds to the lowest R value. The spheres correspond to the solvent regions in the cell. The program has been shown to work successfully for a number of test structures in a variety of space groups. No prior knowledge of the structures is needed, and c.p.u. requirements are extremely modest.

Structure of the catalytic core of the family F xylanase from Pseudomonas fluorescens and identification of the xylopentaose-binding sites.

BACKGROUND: Sequence alignment suggests that xylanases evolved from two ancestral proteins and therefore can be grouped into two families, designated F and G. Family F enzymes show no sequence similarity with any known structure and their architecture is unknown. Studies of an inactive enzyme-substrate complex will help to elucidate the structural basis of binding and catalysis in the family F xylanases. RESULTS: We have therefore determined the crystal structure of the catalytic domain of a family F enzyme, Pseudomonas fluorescens subsp. cellulosa xylanase A, at 2.5 A resolution and a crystallographic R-factor of 0.20. The structure was solved using an engineered catalytic core in which the nucleophilic glutamate was replaced by a cysteine. As expected, this yielded both high-quality mercurial derivatives and an inactive enzyme which enabled the preparation of the inactive enzyme-substrate complex in the crystal. We show that family F xylanases are eight-fold alpha/beta-barrels (TIM barrels) with two active-site glutamates, one of which is the nucleophile and the other the acid-base. Xylopentaose binds to five subsites A-E with the cleaved bond between subsites D and E. Ca2+ binding, remote from the active-site glutamates, stabilizes the structure and may be involved in the binding of extended substrates. CONCLUSIONS: The architecture of P. fluorescens subsp. cellulosa has been determined crystallographically to be a commonly occurring enzyme fold, the eight-fold alpha/beta-barrel. Xylopentaose binds across the carboxy-terminal end of the alpha/beta-barrel in an active-site cleft which contains the two catalytic glutamates.

An unequivocal example of cysteine proteinase activity affected by multiple electrostatic interactions.

The role of electrostatic interactions between the ionizable Asp158 and the active site thiolate-imidazolium ion pair of some cysteine proteinases has been the subject of controversy for some time. This study reports the expression of wild type procaricain and Asp158Glu, Asp158Asn and Asp158Ala mutants from Escherichia coli. Purification of autocatalytically matured enzymes yielded sufficient fully active material for pH (kcat/Km) profiles to be obtained. Use of both uncharged and charged substrates allowed the effects of different reactive enzyme species to be separated from the complications of electrostatic effects between enzyme and substrate. At least three ionizations are detectable in the acid limb of wild type caricain and the Glu and Asn mutants. Only two pKa values, however, are detectable in the acid limb using the Ala mutant. Comparison of pH activity profiles shows that whilst an ionizable residue at position 158 is not essential for the formation of the thiolate-imidazolium ion pair, it does form a substantial part of the electrostatic field responsible for increased catalytic competence. Changing the position of this ionizable group in any way reduces activity. Complete removal of the charged group reduces catalytic competence even further. This work indicates that hydronations distant to the active site are contributing to the electrostatic effects leading to multiple active ionization states of the enzyme.

High-precision direct measurements of (13)CH(4)/(12)CH(4) and (12)CH(3)D/(12)CH(4) ratios in atmospheric methane sources by means of a long-path tunable diode laser absorption spectrometer.

Measurements of (13)CH(4)/(12)CH(4) and (12)CH(3)D/(12)CH(4) ratios in atmospheric methane (CH(4)) sources provide important information about the global CH(4) budget as well as about CH(4) production and consumption processes occurring within the various sources. As an alternative to the conventional mass spectrometer (MS) technique, which requires conversion of CH(4) to CO(2) and H(2), we have developed a tunable diode laser absorption spectrometer (TDLAS), which permits rapid direct measurements of the (13)CH(4)/(12)CH(4) and (12)CH(3)D/(12)CH(4) ratios. An intercomparison between TDLAS and MS techniques for samples from natural wetlands, landfills, and natural gas sources resulted in a mean deviation of Δδ(13)C = 0.44‰ and ΔδD = 5.1‰. In the present system the minimum mixing ratios required are 50 parts in 10(6) by volume (ppmv) CH(4) (sample size 2 µmol CH(4)) for direct δ(13)C measurements and 2000 ppmv (sample size 80 µmol CH(4)) for direct δD measurements. These mixing-ratio limits are adequate for most CH(4) source characterization studies without requiring sample preconcentration.

Factors effecting the thermostability of cysteine proteinases from Carica papaya.

Thermal denaturation of four Carica papaya cysteine proteinases (papain, chymopapain, papaya proteinases 3 and 4) was studied as a function of pH using high-sensitivity differential scanning calorimetry. The ratios of calorimetric enthalpy to Van't Hoff enthalpy suggest that, for all these proteins, denaturation occurs as a non two state process, via an intermediate structure. Differences in the thermal stabilities of the proteinases; chymopapain > papaya proteinase 3 > papain > papaya proteinase 4, were correlated to their amino acid sequence to explain the observations in terms of mobility and specific residue mutation. Three-dimensional structures of papain and papaya proteinase 3 were similarly used to illustrate the influence of atomic mobility on stability.

The segmented anisotropic refinement of monoclinic papain by the application of the rigid-body TLS model and comparison to bovine ribonuclease A.

The anisotropic displacements of selected rigid groups in monoclinic papain have been refined from X-ray diffraction data by application of the rigid-body TLS model. The rigid groups chosen were the aromatic side chains of tryptophan, tyrosine, histidine and phenylalanine, and the planar carboxylic and guanidinium side chains of aspartic acid, glutamic acid, glutamine, asparagine and arginine. The derived translation and libration tensors have been compared with those previously derived for bovine ribonuclease A and provide evidence for different modes and anisotropies of displacement over the two proteins.

Some different strategies of least-squares refinement of a molecule.

Refinements of a macromolecule (ribonuclease-A) based on structure amplitudes, magnitude of F, and structure amplitude squares, magnitude of F2, were carried out and the results compared. Although the conventional R values are higher for the magnitude of F2 refinement, positional parameters from both types of refinement were not significantly different. However, the mean-square displacements from magnitude of F2 refinements were systematically higher than for those using magnitude F. Various resolution windows and weighting schemes were employed during the work. Electron density maps were examined for magnitude of F2 refinements and were very similar to those using magnitude of F in spite of a conventional R factor of 0.29 using all 1.4 A data. While magnitude of F2 refinements may be formally more correct than magnitude of F refinements, there is little evidence that magnitude of F2 refinement is superior provided that a reasonable weighting strategy is adopted.

Frequency modulation spectroscopy at 1.3microm using InGaAsP lasers: a prototype field instrument for atmospheric chemistry research.

Two-tone frequency modulation spectroscopy has been used in conjunction with InGaAsP lasers in the 1.3-microm region to monitor weak water vapor absorptions in a long path White cell. Detection electronics that reduce the effect of Johnson noise are described. The system was capable of detecting optical densities corresponding to <1.7 x 10(-6) in a 1-Hz bandwidth. Factors limiting the difference between observed and shot noise limited performance for these types of laser and condition are discussed.

Segmented anisotropic refinement of bovine ribonuclease A by the application of the rigid-body TLS model.

The anisotropic displacements of selected rigid groups in bovine ribonuclease A have been refined from X-ray diffraction data by the application of the rigid-body TLS model. The rigid groups chosen were the side chains of tyrosine, histidine and phenylalanine and the planar side chains of aspartic acid, glutamic acid, glutamine, asparagine and arginine. The method has also been applied to the co-crystallizing active-site sulfate anion. This has enabled the description of the motion of the above-mentioned side-chain atoms by anisotropic displacement ellipsoids from a 1.45 A refinement. The hydrophobic side groups in the protein core show mainly translational motion, with mean-square librations of 20 deg2 which are similar to those found in some close-packed crystals of small organic molecules. Librational displacements are much more significant in the hydrophilic side groups where their magnitudes can be correlated with solvent accessibility. Large librations of some solvent exposed side chains correspond with the breakdown of a simple TLS model and the existence of multiple orientations of the side groups. The TLS model has also been applied to the whole protein molecule and shows that the average motion is approximately isotropic with little librational character.

X-ray refinement study on the binding of cytidylic acid (2'-CMP) to ribonuclease A.

The X-ray structure of the inhibitor complex of bovine ribonuclease A with cytidylic acid (2'-CMP) has been determined at 2.3 A (1 A = 0.1 nm) resolution and refined by restrained least-squares refinement to R = 0.132 for 5650 reflections. Incorporation of the inhibitor molecule has occurred with little disturbance of the protein main-chain atoms, although significant displacement of some side-chain atoms has occurred, particularly in the region of the active site. The binding of 2'-CMP to ribonuclease A is different from that of the related cytidine-N(3)-oxide 2'-phosphate, which has an extra oxygen on N(3) of the cytidine base. The PO4(2-) group is held by hydrogen bond interactions to the side-groups of His 12, Glu 11 and His119. Thr45 is involved in stabilizing the enzyme-ligand complex by forming hydrogen bond interactions between O(gamma) and the pyrimidine base N(3) atom and between the main-chain N(45) and O(2) of the base. Phe120 is much closer to the inhibitor than in the cytidine N(3)-oxide 2'-phosphate structure.