B.W. Dijkstra

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)

Detergent organisation in crystals of monomeric outer membrane phospholipase A.

The structure of the detergent in crystals of outer membrane phospholipase A (OMPLA) has been determined using neutron diffraction contrast variation. Large crystals were soaked in stabilising solutions, each containing a different H(2)O/D(2)O contrast. From the neutron diffraction at five contrasts, the 12 A resolution structure of the detergent micelle around the protein molecule was determined. The hydrophobic beta-barrel surfaces of the protein molecules are covered by rings of detergent. These detergent belts are fused to neighbouring detergent rings forming a continuous three-dimensional network throughout the crystal. The thickness of the detergent layer around the protein varies from 7-20 A. The enzymes active site is positioned just outside the hydrophobic detergent zone and is thus in a proper location to catalyse the hydrolysis of phospholipids in a natural membrane. Although the dimerisation face of OMPLA is covered with detergent, the detergent density is weak near the exposed polar patch, suggesting that burying this patch in the enzymes dimer interface may be energetically favourable. Furthermore, these results indicate a crucial role for detergent coalescence during crystal formation and contribute to the understanding of membrane protein crystallisation.

Protein engineering of cyclodextrin glycosyltransferase from Bacillus circulans strain 251

Abstract The 3D-structure of CGTase from Bacillus circulans strain 251 has been determined at 2.0xa0A resolution, allowing a detailed analysis of structure-function relationships of this protein. The catalytic mechanism of the CGTase cyclization reaction was studied by constructing various mutants using site-directed mutagenesis. 3D-structures of these mutant proteins have been obtained and further analysed.

Structural investigations of the active-site mutant Asn156Ala of outer membrane phospholipase A: Function of the Asn-His interaction in the catalytic triad

Outer membrane phospholipase A (OMPLA) from Escherichia coli is an integral‐membrane enzyme with a unique His–Ser–Asn catalytic triad. In serine proteases and serine esterases usually an Asp occurs in the catalytic triad; its role has been the subject of much debate. Here the role of the uncharged asparagine in the active site of OMPLA is investigated by structural characterization of the Asn156Ala mutant. Asparagine 156 is not involved in maintaining the overall active‐site configuration and does not contribute significantly to the thermal stability of OMPLA. The active‐site histidine retains an active conformation in the mutant notwithstanding the loss of the hydrogen bond to the asparagine side chain. Instead, stabilization of the correct tautomeric form of the histidine can account for the observed decrease in activity of the Asn156Ala mutant.

Non-covalent binding of the heavy atom compound [Au(CN)2]− at the halide binding site of haloalkane dehalogenase from Xanthobacter autotrophicus GJ10

The Na[Au(CN)2] heavy atom derivative contributed considerably to the successful elucidation of the crystal structure of haloalkane dehalogenase isolated from Xanthobacter autotrophicus GJ10. The gold cyanide was located in an internal cavity of the enzyme, which also contains the catalytic residues. Refinement of the dehalogenase‐gold cyanide complex at 0.25 nm to an R‐factor of 16.7% demonstrates that the heavy atom molecule binds non‐covalently between two tryptophan residues pointing into the active site cavity. At this same site also chloride ions can be bound. Therefore, inhibition of dehalogenase activity by the Au(CN)2 − presumably occurs by competition for the same binding site as substrates.

Structural investigations of the active-site mutant Asn156Ala of outer membrane phospholipase A

Outer membrane phospholipase A (OMPLA) from Escherichia coliis an integral-membrane enzyme with a unique His–Ser–Asn catalytic triad. In serine proteases and serine esterases usually an Asp occurs in the catalytic triad; its role has been the subject of much debate. Here the role of the uncharged asparagine in the active site of OMPLA is investigated by structural characterization of the Asn156Ala mutant. Asparagine 156 is not involved in maintaining the overall active-site configuration and does not contribute significantly to the thermal stability of OMPLA. The active-site histidine retains an active conformation in the mutant notwithstanding the loss of the hydrogen bond to the asparagine side chain. Instead, stabilization of the correct tautomeric form of the histidine can account for the observed decrease in activity of the Asn156Ala mutant.

Structural and Functional Consequences of Engineering the High Alkaline Serine Protease PB92

The ability to remove proteinaceous fabric stains made the alkaline proteases powerful tools in the hands of detergent manufacturers. The application of proteases in detergents requires that the proteases are stable and active in the presence of typical detergent ingredients such as surfactants, builders, bleaching agents, bleach activators, fillers, fabric softeners, various formulation aids, etc. To meet the high alkaline conditions in heavy duty powder detergents, extracellular Bacillus serine proteases have been extensively screened for good detergent stability and a high wash performance at this pH. In such a screening an alkalophilic Bacillus strain, PB92, was isolated1 which produces a serine protease with optimal performance in the pH range 10 to 12. The gene encoding this high-alkaline protease PB92 has been cloned and characterized2. The mature protease has a molecular weight of 26,900 and consists of 269 amino acids. A high sequence homology exists with protease Yab3 (83%) which is also produced by an alkalophilic Bacillus while moderate homology is found with the well known serine proteases from Bacillus amyloliquefaciens (subtilisin BPN’/NOVO, 59%)4,5, B. licheniformis (subtilisin Carlsberg, 60%)6,7, Tritirachium album Limber (proteinase-K, 34%)8,9, and Thermoactinomyces vulgaris (thermitase, 47%)10. In a recent review Siezen et al.11 proposed the name’subtilases’ for the group of serine proteases which share amino acid sequence homology with BPN’/NOVO in order to distinguish them from the (chymo)trypsin related serine proteases.

The active site of phospholipase A2

Inhibition of enzymes is usually brought about by reagents which attack essential functional groups in the active site or by blocking the entrance to the active site. If one for example reacts the essential His 48 in phospholipase A2 with an acylating reagent such as p-bromophenacyl-bromide all activity is lost. However the activity of phospholipase A2 towards phospholipid micelles depends not only on the integrity of the active site itself, but also on the mobility of parts of the polypeptide chain which do not immediately belong to the active site. This became apparent from a comparison of the structure of native pancreatic phospholipase A2 and its precursor. This mobility is closely linked to the condition of the N-terminus.