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B W Dijkstra

Publications and source records attributed to B W Dijkstra.

124 records · Page 7Linked to original sources
PubMed1989

Enhanced activity and altered specificity of phospholipase A2 by deletion of a surface loop.

Protein engineering and x-ray crystallography have been used to study the role of a surface loop that is present in pancreatic phospholipases but is absent in snake venom phospholipases. Removal of residues 62 to 66 from porcine pancreatic phospholipase A2 does not change the binding constant for micelles significantly, but it improves catalytic activity up to 16 times on micellar (zwitterionic) lecithin substrates. In contrast, the decrease in activity on negatively charged substrates is greater than fourfold. A crystallographic study of the mutant enzyme shows that the region of the deletion has a well-defined structure that differs from the structure of the wild-type enzyme. No structural changes in the active site of the enzyme were detected.

Amino Acid Sequence↗
PubMed1989

X-ray structure of lipoamide dehydrogenase from Azotobacter vinelandii determined by a combination of molecular and isomorphous replacement techniques.

The crystal structure of lipoamide dehydrogenase from Azotobacter vinelandii has been determined by a combination of molecular replacement and isomorphous replacement techniques yielding eventually a good-quality 2.8 A electron density map. Initially, the structure determination was attempted by molecular replacement procedures alone using a model of human glutathione reductase, which has 26% sequence identity with this bacterial dehydrogenase. The rotation function yielded the correct orientation of the model structure both when the glutathione reductase dimer and monomer were used as starting model. The translation function could not be solved, however. Consequently, data for two heavy-atom derivatives were collected using the Hamburg synchotron facilities. The derivatives had several sites in common, which was presumably a major reason why the electron density map obtained by isomorphous information alone was of poor quality. Application of solvent flattening procedures cleaned up the map considerably, however, showing clearly the outline of the lipoamide dehydrogenase dimer, which has a molecular weight of 100,000. Application of the "phased translation function", which combines the phase information of both isomorphous and molecular replacement, led to an unambiguous determination of the position of the model structure in the lipoamide dehydrogenase unit cell. The non-crystallographic 2-fold axis of the dimer was optimized by several cycles of constrained-restrained least-squares refinement and subsequently used for phase improvement by 2-fold density averaging. After ten cycles at 3.5 A, the resolution was gradually extended to 2.8 A in another 140 cycles. The 2.8 A electron density distribution obtained in this manner was of much improved quality and allowed building of an atomic model of A. vinelandii lipoamide dehydrogenase. It appears that in the orthorhombic crystals used each dimer is involved in contacts with eight surrounding dimers, leaving unexplained why the crystals are rather fragile. Contacts between subunits within one dimer, which are quite extensive, can be divided into two regions separated by a cavity. In one of the contact regions, the level of sequence identity with glutathione reductase is very low but it is quite high in the other. The folding of the polypeptide chain in each subunit is quite similar to that of glutathione reductase, as is the extended conformation of the co-enzyme FAD.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1989

Use of electron microscopy in the examination of lattice defects in crystals of alcohol oxidase.

Alcohol oxidase, purified from the yeast Hansenula polymorpha, was crystallized in vitro for the purpose of determining its structure at atomic resolution by X-ray diffraction methods. The crystals obtained yielded only extremely weak diffraction patterns: the maximal resolution observed was in the best case 6 A. Electron microscopy of thin sections indicated that most crystals showed lattice defects which might explain the poor diffraction patterns: most surprising was the appearance of large holes interrupting an otherwise regular lattice in one of the crystal forms examined. Our results indicate that transmission electron microscopy is a suitable tool for the inspection of crystals to be used in X-ray crystallography. The method allows rapid determination of lattice defects and enables optimization of crystallization conditions.

Alcohol Oxidoreductases↗
PubMed1988

Crystallization of haloalkane dehalogenase from Xanthobacter autotrophicus GJ10.

Haloalkane dehalogenases are enzymes that release chloride or bromide from n-halogenated alkanes. X-ray quality crystals of haloalkane dehalogenase from the 1,2-dichloroethane-degrading bacterium Xanthobacter autotrophicus GJ10 have been grown at room temperature from 64% saturated ammonium sulfate solutions (pH 6.2 to 6.4). The crystals diffract in the X-ray beam to at least 2.4 A resolution (1 A = 0.1 nm). Their space group is P2(1)2(1)2, with cell dimensions a = 94.1 A, b = 72.8 A, c = 41.4 A and alpha = beta = gamma = 90 degrees. There is one monomer (molecular weight 36,000) per asymmetric unit.

Gram-Negative Aerobic Bacteria↗
PubMed1988

Crystal structure of bovine pancreatic phospholipase A2 covalently inhibited by p-bromo-phenacyl-bromide.

Bovine pancreatic phospholipase A2 covalently inhibited by p-bromo-phenacyl-bromide was crystallized from 50% (v/v) 2-methyl-2,4-pentanediol. The space group was P3(1)21 with cell dimensions a = b = 46.73 A and c = 102.5 A (1 A = 0.1 nm). Diffraction data were collected by oscillation photography from one single crystal of dimensions 0.2 mm x 0.2 mm x 0.2 mm. The crystal structure was determined to a resolution of 2.5 A by crystallographic refinement of a starting model, which consisted of native bovine pancreatic phospholipase A2 positioned and oriented in the P3(1)21 cell as in the bovine pro-phospholipase A2. The crystallographic R-factor decreased from 0.378 to 0.197 after 70 refinement cycles. For the greater part the three-dimensional structure was very similar to that of native phospholipase. The inhibitor group shows up clearly. However, as in solution, there is no calcium ion bound any more in the active site, and this causes a significant conformational change in the loop from residue 59 to 73. This loop is remote from the calcium binding site. Interestingly, this is the same loop that also shows different conformations in other phospholipase A2 molecules. The inhibitor molecule has hydrophobic interactions with Phe5 and Cys45. Rational design of specific and potent inhibitors of phospholipase A2 catalysis is discussed on the basis of the present three-dimensional structure.

Acetophenones↗
PubMed1988

Model building of disulfide bonds in proteins with known three-dimensional structure.

As an aid in the selection of sites in a protein where a disulfide bond might be engineered, a computer program has been developed. The algorithm starts with the generation of C beta positions from the N, C alpha and C atom coordinates available from a three-dimensional model. A first set of residue pairs that might form a disulfide bond is selected on the basis of C beta-C beta distances between residues. Then, for each residue in this set, S gamma positions are generated, which satisfy the requirement that, with ideal values for the C alpha-C beta and C beta-S gamma bond lengths and for the bond angle at C beta, the distance between S gamma of residue 1 and C beta of residue 2 in a pair (determined by the bond angle at S gamma 2) is at, or very close to its ideal value. Usually two acceptable S gamma positions are found for each half cystine, resulting in up to four different conformations for the disulfide bond. Finally, these conformations are subjected to an energy minimization procedure to remove large deviations from ideal geometry and their final energies are calculated. User input determines which final conformations are energetically acceptable. These conformations are written to a file to allow further analysis and e.g. inspection on a computer graphics device.

Algorithms↗
PubMed1985

A comparison of the crystal structures of phospholipase A2 from bovine pancreas and Crotalus atrox venom.

The refined high resolution crystal structure of the bovine phospholipase A2 was compared with its counterpart from the venom of Crotalus atrox, the western diamondbacked rattlesnake. The strong similarity in their backbone conformations forms the basis of a common numbering system for the amino acid sequence. The three common major helices and much of the extended chain form a nearly identical "homologous core" structure. The variations in conformation usually arise from deletions/insertions or en bloc shifts of structural units. The exception to this is part of the highly conserved calcium-binding loop; however, this is to be expected as 1) there is no calcium ion sequestered in the venom dimer as there is in the case of the bovine enzyme and 2) two side chains in that segment form dimer-stabilizing interactions between the subunits of the C. atrox enzyme. The absolutely conserved catalytic network of hydrogen-bonded side chains formed by His 48, Tyr 52, Tyr 73, and Asp 99, as well as the hydrophobic wall that shields it, are virtually superimposable in the two structures. However, the details of the structural relationship between the amino terminus and the catalytic network differ in the two species and the ordered water molecules thought to be either functionally or structurally important in the pancreatic enzymes are not found in the crystal structure of the phospholipase A2 from C. atrox. The most striking difference from a functional standpoint is the fact that the surface depression in the region of the catalytic network that has been commonly considered the active site is shielded substantially in forming the intersubunit contact surface of the dimeric venom enzyme.

Animals↗
PubMed1984

Role of the N-terminus in the interaction of pancreatic phospholipase A2 with aggregated substrates. Properties and crystal structure of transaminated phospholipase A2.

A free N-terminal alpha-NH3+ group is absolutely required for full catalytic activity of phospholipase A2 on aggregated substrates. To elucidate how this alpha-NH3+ group triggers catalytic activity, we specifically transaminated this group in various pancreatic phospholipases A2. Porcine, porcine iso-, equine, human, ovine, and bovine phospholipases A2 all loose catalytic activity on micellar substrates due to the inability of the transaminated proteins to bind to neutral micellar substrate analogues, as was found for the zymogens. Loss of activity is pseudo first order, the rate constants being different for the enzymes studied. The transaminated phospholipases A2 have an intact active site, as catalytic activities on monomeric substrates are comparable to those of the respective zymogens. The X-ray structure of transaminated bovine phospholipase A2 at 2.1-A resolution shows that the N-terminal region and the sequence 63-72 in this protein are more flexible than in the native enzyme. Also, in this respect, the transaminated enzyme very much resembles the zymogen structure. In good agreement with this, it was found by photochemically induced dynamic nuclear polarization 1H NMR that aromatic resonances of Trp-3 and Tyr-69 are affected by transamination. In addition, fluorescence spectroscopy of the unique Trp-3 in transaminated bovine phospholipase A2 revealed a red shift of the emission maximum indicative of a more polar environment of Trp-3 in the transaminated phospholipase A2 as compared to the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1983

Polypeptide chains with similar amino acid sequences but a distinctly different conformation. Bovine and porcine phospholipase A2.

The primary structures of bovine and porcine pancreatic phospholipase A2 differ only by about 15%. Nevertheless, a 12 residue loop, with only one substitution (Val leads to Phe) has a quite different conformation, whereas the rest of the molecules have a very similar folding indeed. From this observation it is concluded that prediction of a 3-dimensional structure on the basis of sequence similarity of short segments alone might give erroneous results.

Amino Acid Sequence↗
PubMed1983

Structure of porcine pancreatic phospholipase A2 at 2.6 A resolution and comparison with bovine phospholipase A2.

The previously published three-dimensional structure of porcine pancreatic prophospholipase A2 at 3 A resolution was found to be incompatible with the structures of bovine phospholipase A2 and bovine prophospholipase A2. This was unexpected because of the very homologous amino acid sequences of these enzymes. Therefore, the crystal structure of the porcine enzyme was redetermined using molecular replacement methods with bovine phospholipase as the parent model. The structure was crystallographically refined at 2.6 A resolution by fast Fourier transform and restrained least-squares procedures to an R-factor of 0.241. The crystals appeared to contain phospholipase A2 and not prophospholipase A2. Apparently the protein is slowly converted under the crystallization conditions employed. Our investigation shows that, in contrast to the previous report, the three-dimensional structure of porcine phospholipase A2 is very similar to that of bovine phospholipase A2, including the active site. Smaller differences were observed in some residues involved in the binding of aggregated substrates. However, an appreciable conformational difference is in the loop 59 to 70, where a single substitution at position 63 (bovine Val leads to porcine Phe) causes a complete rearrangement of the peptide chain. In addition to the calcium ion in the active site, a second calcium ion is present in the crystals; this is located on a crystallographic 2-fold axis and stabilizes the interaction between two neighbouring molecules.

Amino Acid Sequence↗
PubMed1981

Active site and catalytic mechanism of phospholipase A2.

The esterolytic enzyme phospholipase A2 specificially splits the 2-acyl linkage of phosphoglycerides in a calcium-dependent reaction. In the pancreas the enzyme occurs as a zymogen which is activated on secretion into the duodenal tract by the removal of seven amino acid residues from the N terminus by trypsin. Having refined our X-ray analysis of the crystal structure of bovine pancreatic phospholipase A2 from 2.4 A (ref. 4) to 1.7 A resolution, we now describe how the structure of the molecule may account for the specificity of the enzyme and for the sudden and dramatic change in activity when the substrate concentration passes the critical micelle concentration.

Animals↗
PubMed1980

Methylation of histidine-48 in pancreatic phospholipase A2. Role of histidine and calcium ion in the catalytic mechanism.

It is known that His-48 is part of the active center in pancreatic phospholipase. To further elucidate the role of histidine-48 in the active center of pancreatic phospholipase A2, we have modified the enzyme with a number of bromo ketones and methyl benzenesulfonates. Rapid methylation occurred with methyl p-nitrobenzenesulfonate. Methylated phospholipase shows total loss of enzymatic activity whereas binding of substrate and the cofactor Ca2+ remains intact. Amino acid analysis of methylated equine phospholipase showed the loss of the single molecule of histidine and the formation of one molecule of 2-amino-3-(1-methyl-5-imidazolyl)propanoic acid (1-methylhistidine). Equine phospholipase was also modified by [13C]methyl p-nitrobenzenesulfonate and the methylated enzyme was studied by 13C NMR. The results indicate that the proton on the nitrogen in position 3 of the imidazole ring is involved in a strong interaction with a buried carboxylate group, thereby hindering rotation of the imidazole ring, and that the nitrogen in position 1 is involved in catalysis. These data are in full agreement with the three-dimensional structure at 1.7-A resolution of bovine pancreatic phospholipase. A catalytic mechanism is proposed in which a water molecule which is close to the nitrogen at position 1 of the imidazole ring of the Asp-99-His-48 couple acts as the nucleophile. A comparison is made between phospholipase A2 and the serine esterases.

Animals↗
PubMedDate not supplied

Crystallization and preliminary X-ray analysis of a lipase from Staphylococcus hyicus.

Single cyrstals of a lipase from Staphylococcus hyicus have been obtained using a combination of 18 to 24% dimethylsulfoxide and 10% isopropanol as a precipitant. The crystals grow at 4 degrees C in 2-3 months. They belong to the orthorhombic space group P212121 with a = 73.31 A, b = 77.96 A, and c = 169.81 A, with two protein molecules per asymmetrical unit. The crystals diffract to at least 2.8 A resolution and are suitable for an X-ray structure analysis.

Crystallization↗