Atomic resolution structure of the double mutant (K53,56M) of bovine pancreatic phospholipase A2.

The structure of the double mutant K53,56M has previously been refined at 1.9 A resolution using room-temperature data. The present paper reports the crystal structure of the same mutant K53,56M refined against 1.1 A data collected using synchrotron radiation. A total of 116 main-chain atoms from 29 residues and 44 side chains are modelled in alternate conformations. Most of the interfacial binding residues are found to be disordered and alternate conformations could be recognized. The second calcium ion-binding site residue Glu92 adopts two alternate conformations. The minor and major conformations of Glu92 correspond to the second calcium ion bound and unbound states.

Anomalous signal of solvent bromides used for phasing of lysozyme.

The anomalous signal of bromide ions, present in the crystal structure of tetragonal hen egg-white lysozyme through the substitution of NaCl by NaBr in the crystallization medium, was used for phasing of X-ray data collected to 1.7 A resolution with a wavelength near the absorption edge of bromine. Phasing of a single wavelength data set, based purely on anomalous deltaf " contribution, led to easily interpretable electron density, equivalent to the complete multiwavelength anonalous dispersion phasing based on four-wavelength data. The classic small-structure direct methods program SHELXS run against all anomalous differences gave a successful solution of six highest peaks corresponding to six bromide ions in the structure with data limited up to a resolution of 3.5 A. Interpretable maps were obtained at a resolution up to 3.0 A using programs MLPHARE and DM. Bromide ions occupy well ordered positions at the protein surface. Phasing based on the single wavelength signal of anomalous scatterers introduced into the ordered solvent shell can be proposed as a tool for solving structures of well diffracting crystals.

Can anomalous signal of sulfur become a tool for solving protein crystal structures?

A general method for solving the phase problem from native crystals of macromolecules has long eluded structural biology. For well diffracting crystals this goal can now be achieved, as is shown here, thanks to modern data collection techniques and new statistical phasing algorithms. Using solely a native crystal of tetragonal hen egg-white lysozyme, a protein of 14 kDa molecular mass, it was possible to detect the positions of the ten sulfur and seven chlorine atoms from their anomalous signal, and proceed from there to obtain an electron-density map of very high quality.

Catalysis and specificity in enzymatic glycoside hydrolysis: a 2,5B conformation for the glycosyl-enzyme intermediate revealed by the structure of the Bacillus agaradhaerens family 11 xylanase.

BACKGROUND: The enzymatic hydrolysis of glycosides involves the formation and subsequent breakdown of a covalent glycosyl-enzyme intermediate via oxocarbenium-ion-like transition states. The covalent intermediate may be trapped on-enzyme using 2-fluoro-substituted glycosides, which provide details of the intermediate conformation and noncovalent interactions between enzyme and oligosaccharide. Xylanases are important in industrial applications - in the pulp and paper industry, pretreating wood with xylanases decreases the amount of chlorine-containing chemicals used. Xylanases are structurally similar to cellulases but differ in their specificity for xylose-based, versus glucose-based, substrates. RESULTS: The structure of the family 11 xylanase, Xyl11, from Bacillus agaradhaerens has been solved using X-ray crystallography in both native and xylobiosyl-enzyme intermediate forms at 1.78 A and 2.0 A resolution, respectively. The covalent glycosyl-enzyme intermediate has been trapped using a 2-fluoro-2-deoxy substrate with a good leaving group. Unlike covalent intermediate structures for glycoside hydrolases from other families, the covalent glycosyl-enzyme intermediate in family 11 adopts an unusual 2,5B conformation. CONCLUSIONS: The 2,5B conformation found for the alpha-linked xylobiosyl-enzyme intermediate of Xyl11, unlike the 4C1 chair conformation observed for other systems, is consistent with the stereochemical constraints required of the oxocarbenium-ion-like transition state. Comparison of the Xyl11 covalent glycosyl-enzyme intermediate with the equivalent structure for the related family 12 endoglucanase, CelB, from Streptomyces lividans reveals the likely determinants for substrate specificity in this clan of glycoside hydrolases.

Atomic resolution (0.97 A) structure of the triple mutant (K53,56,121M) of bovine pancreatic phospholipase A2.

The enzyme phospholipase A2 catalyzes the hydrolysis of the sn-2 acyl chain of phospholipids, forming fatty acids and lysophospholipids. The crystal structure of a triple mutant (K53,56,121M) of bovine pancreatic phospholipase A2 in which the lysine residues at positions 53, 56 and 121 are replaced recombinantly by methionines has been determined at atomic resolution (0.97 A). The crystal is monoclinic (space group P2), with unit-cell parameters a = 36.934, b = 23.863, c = 65.931 A, beta = 101.47 degrees. The structure was solved by molecular replacement and has been refined to a final R factor of 10.6% (Rfree = 13.4%) using 63,926 unique reflections. The final protein model consists of 123 amino-acid residues, two calcium ions, one chloride ion, 243 water molecules and six 2-methyl-2,4-pentanediol molecules. The surface-loop residues 60-70 are ordered and have clear electron density.

Crystallisation and preliminary analysis of glucose isomerase from Streptomyces albus.

The glucose isomerase of Streptomyces albus has been crystallised from a dilute solution of magnesium chloride buffered at a pH of 6.8-7.0. The crystals are in the space group I222 with cell dimensions a = 93.9 A, b = 99.5 A and c = 102.9 A. There is one monomer of the tetrameric molecule per asymmetric unit of the crystal and the packing density is 2.93 A3.Da-1. The tetramer sits on the 222 symmetry point of the crystal. Native data have been recorded to a resolution of 1.9 A and the crystals diffract to about 1.5 A. The alpha-carbon coordinates of the Arthrobacter glucose isomerase and the backbone coordinates of the S. olivochromogenes enzyme determined by other groups have been oriented in the present cell. The structure is currently being refined. The binding of several metal ions to the two metal sites has been analysed.

Novel approach to phasing proteins: derivatization by short cryo-soaking with halides.

A quick (less than 1 min) soak of protein crystals in a cryo-solution containing bromide or iodide anions leads to incorporation of these anomalous scatterers into the ordered solvent region around the protein molecules. These halide anions provide a convenient way of phasing through their anomalous scattering signal: bromides using multiwavelength anomalous dispersion (MAD) and bromides and/or iodides using single-wavelength anomalous dispersion (SAD) or single isomorphous replacement with anomalous scattering (SIRAS) methods. This approach has been tested successfully on four different proteins and has been used to solve the structure of a new protein of molecular weight 30 kDa.

Fusarium oxysporum trypsin at atomic resolution at 100 and 283 K: a study of ligand binding.

The X-ray structure of F. oxysporum trypsin has been determined at atomic resolution, revealing electron density in the binding site which was interpreted as a peptide bound in the sites S1, S2 and S3. The structure, which was initially determined at 1.07 A resolution and 283 K, has an Arg in the S1 specificity pocket. The study was extended to 0.81 A resolution at 100 K using crystals soaked in Arg, Lys and Gln to study in greater detail the binding at the S1 site. The electron density in the binding site was compared between the different structures and analysed in terms of partially occupied and overlapping components of peptide, solvent water and possibly other chemical moieties. Arg-soaked crystals reveal a density more detailed but similar to the original structure, with the Arg side chain visible in the S1 pocket and residual peptide density in the S2 and S3 sites. The density in the active site is complex and not fully interpreted. Lys at high concentrations displaces Arg in the S1 pocket, while some main-chain density remains in sites S2 and S3. Gln has been shown not to bind. The free peptide in the S1-S3 sites binds in a similar way to the binding loop of BPTI or the inhibitory domain of the Alzheimer's beta-protein precursor, with some differences in the S1 site.

Structure and function of Humicola insolens family 6 cellulases: structure of the endoglucanase, Cel6B, at 1.6 A resolution.

Cellulases are traditionally classified as either endoglucanases or cellobiohydrolases on the basis of their respective catalytic activities on crystalline cellulose, which is generally hydrolysed more efficiently only by the cellobiohydrolases. On the basis of the Trichoderma reesei cellobiohydrolase II structure, it was proposed that the active-site tunnel of cellobiohydrolases permitted the processive hydrolysis of cellulose, whereas the corresponding endoglucanases would display open active-site clefts [Rouvinen, Bergfors, Teeri, Knowles and Jones (1990) Science 249, 380-386]. Glycoside hydrolase family 6 contains both cellobiohydrolases and endoglucanases. The structure of the catalytic core of the family 6 endoglucanase Cel6B from Humicola insolens has been solved by molecular replacement with the known T. reesei cellobiohydrolase II as the search model. Strangely, at the sequence level, this enzyme exhibits the highest sequence similarity to family 6 cellobiohydrolases and displays just one of the loop deletions traditionally associated with endoglucanases in this family. However, this enzyme shows no activity on crystalline substrates but a high activity on soluble substrates, which is typical of an endoglucanase. The three-dimensional structure reveals that the deletion of just a single loop of the active site, coupled with the resultant conformational change in a second 'cellobiohydrolase-specific' loop, peels open the active-site tunnel to reveal a substrate-binding groove.

A redetermination of the structure of the triple mutant (K53,56,120M) of phospholipase A2 at 1.6 A resolution using sulfur-SAS at 1.54 A wavelength.

The crystal structure of the triple mutant K53,56,120M of bovine pancreatic phospholipase A(2) has been redetermined using sulfur single-wavelength anomalous scattering. The synchrotron data were collected at lambda = 1.54 A and the crystal diffracted to 1.6 A resolution. The program SOLVE was used to locate the heavy atoms and to estimate the initial phases and the resulting map was then subjected to RESOLVE. The output of 455 non-H atoms, including 12 S atoms, one calcium ion and one chloride ion, were then subjected to ARP/wARP followed by REFMAC. With the improved phases, the automatic model building successfully built more than 85% of the 123 residues, excluding the N- and C-terminal residues. The final crystallographic R factor is 17.7% (R(free) = 21.7%). The refined model consists of 954 non-H protein atoms, 165 water O atoms, three 2-methyl-2,4-pentanediol (MPD) molecules, one calcium ion and one chloride ion. The present work is yet another example that shows the utility of single-wavelength anomalous scattering data for solving a protein structure.

Structure of inhibited trypsin from Fusarium oxysporum at 1.55 A.

The structure of trypsin from the fungus Fusarium oxysporum has been refined at 1.55 A resolution by restrained least-squares minimization to an R-factor of 14.4%. The data were recorded from a single-crystal on the X31 beamline at EMBL, Hamburg, using a locally developed image-plate scanner. The final model consists of 1557 protein atoms, 400 water molecules, one molecule of isopropanol and one monoisopropyl phosphoryl inhibitor group covalently bound to the catalytic Ser195. Comparison of the structure with bovine trypsin reveals significant differences in the active site and suggests a possible explanation for the difference in substrate specificity between the two enzymes. In F. oxysporum trypsin the specificity pocket is larger than in bovine trypsin. This explains the preference of F. oxysporum trypsin for the bulkier arginine over lysine and the reverse preference in bovine trypsin. The binding cavity on the C-terminal side of the substrate is more restricted in F. oxysporum trypsin than in mammalian and Streptomyces griseus trypsins, which explains the relative inactivity of F. oxysporum trypsin towards peptide-pNA substrate analogues as an unfavourable steric interaction between the side of the binding cavity and the para-nitroanilino group of peptide-pNA. The observed restriction of the binding cavity does not lead to a reduced catalytic activity compared to other trypsins.

Snapshots along an enzymatic reaction coordinate: analysis of a retaining beta-glycoside hydrolase.

The enzymatic hydrolysis of O-glycosidic linkages is one of the most diverse and widespread reactions in nature and involves a classic "textbook" enzyme mechanism. A multidisciplinary analysis of a beta-glycoside hydrolase, the Cel5A from Bacillus agaradhaerens, is presented in which the structures of each of the native, substrate, covalent-intermediate, and product complexes have been determined and their interconversions analyzed kinetically, providing unprecedented insights into the mechanism of this enzyme class. Substrate is bound in a distorted 1S3 skew-boat conformation, thereby presenting the anomeric carbon appropriately for nucleophilic attack as well as satisfying the stereoelectronic requirements for an incipient oxocarbenium ion. Leaving group departure results in the trapping of a covalent alpha-glycosyl-enzyme intermediate in which the sugar adopts an undistorted 4C1 conformation. Finally, hydrolysis of this intermediate yields a product complex in which the sugar is bound in a partially disordered mode, consistent with unfavorable interactions and low product affinity.

The sequence and X-ray structure of the trypsin from Fusarium oxysporum.

The trypsin from Fusarium oxysporum is equally homologous to trypsins from Streptomyces griseus, Streptomyces erythraeus and to bovine trypsin. A DFP (diisopropylfluorophosphate) inhibited form of the enzyme has been crystallized from 1.4 M Na2SO4, buffered with citrate at pH 5.0-5.5. The crystals belong to space group P2(1) with cell parameters a = 33.43 A, b = 67.65 A, c = 39.85 A and beta = 107.6 degrees. There is one protein molecule in the asymmetric unit. X-ray diffraction data to a resolution of 1.8 A were collected on film using synchrotron radiation. The structure was solved by molecular replacement using models of bovine and S. griseus trypsins and refined to an R-factor of 0.141. The overall fold is similar to other trypsins, with some insertions and deletions. There is no evidence of the divalent cation binding sites seen in other trypsins. The covalently bound inhibitor molecule is clearly visible.

Structure of the Bacillus agaradherans family 5 endoglucanase at 1.6 A and its cellobiose complex at 2.0 A resolution.

The enzymatic degradation of cellulose, by cellulases, is not only industrially important in the food, paper, and textile industries but also a potentially useful method for the environmentally friendly recycling of municipal waste. An understanding of the structural and mechanistic requirements for the hydrolysis of the beta-1,4 glycosidic bonds of cellulose is an essential prerequisite for beneficial engineering of cellulases for these processes. Cellulases have been classified into 13 of the 62 glycoside hydrolase families [Henrissat, B., and Bairoch, A. (1996) Biochem J. 316, 695-696]. The structure of the catalytic core of the family 5 endoglucanase, Ce15A, from the alkalophilic Bacillus agaradherans has been solved by multiple isomorphous replacement at 1.6 A resolution. Ce15A has the (alpha/beta)8 barrel structure and signature structural features typical of the grouping of glycoside hydrolase families known as clan GH-A, with the catalytic acid/base Glu 139 and nucleophile Glu 228 on barrel strands beta 4 and beta 7 as expected. In addition to the native enzyme, the 2.0 A resolution structure of the cellobiose-bound form of the enzyme has also been determined. Cellobiose binds preferentially in the -2 and -3 subsites of the enzyme. Kinetic studies on the isolated catalytic core domain of Ce15A, using a series of reduced cellodextrins as substrates, suggest approximately five to six binding sites, consistent with the shape and size of the cleft observed by crystallography.

X-ray structure of Novamyl, the five-domain "maltogenic" alpha-amylase from Bacillus stearothermophilus: maltose and acarbose complexes at 1.7A resolution.

The three-dimensional structure of the Bacillus stearothermophilus "maltogenic" alpha-amylase, Novamyl, has been determined by X-ray crystallography at a resolution of 1.7 A. Unlike conventional alpha-amylases from glycoside hydrolase family 13, Novamyl exhibits the five-domain structure more usually associated with cyclodextrin glycosyltransferase. Complexes of the enzyme with both maltose and the inhibitor acarbose have been characterized. In the maltose complex, two molecules of maltose are found in the -1 to -2 and +2 to +3 subsites of the active site, with two more on the C and E domains. The C-domain maltose occupies a position identical to one previously observed in the Bacillus circulans CGTase structure [Lawson, C. L., et al. (1994) J. Mol. Biol. 236, 590-600], suggesting that the C-domain plays a genuine biological role in saccharide binding. In the acarbose-maltose complex, the tetrasaccharide inhibitor acarbose is found as an extended hexasaccharide species, bound in the -3 to +3 subsites. The transition state mimicking pseudosaccharide is bound in the -1 subsite of the enzyme in a 2H3 half-chair conformation, as expected. The active site of Novamyl lies in an open gully, fully consistent with its ability to perform internal cleavage via an endo as opposed to an exo activity.

Crystallization and preliminary X-ray crystallographic analysis of a Trichoderma reesei beta-mannanase from glycoside hydrolase family 5.

Crystals of the catalytic core domain of a Trichoderma reesei beta-mannanase belonging to glycoside hydrolase family 5 have been grown by the sitting-drop method at room temperature using ammonium sulfate as precipitant. The crystals grow as thin colourless plates and belong to space group P21, with unit-cell parameters a = 50.0, b = 54.3, c = 60.2 A, beta = 111.3 degrees, and have a single monomer of mannanase in the asymmetric unit. Native data to 2.0 A resolution have been collected at room temperature using synchrotron radiation. Data for a platinum derivative have been collected to 1.65 A at 110 K in a very short time at the CCLRC Daresbury synchrotron source, using a charge-coupled device (CCD) as detector.