Ulrike Wagner

Improved molecular replacement by density- and energy-guided protein structure optimization

Molecular replacement procedures, which search for placements of a starting model within the crystallographic unit cell that best account for the measured diffraction amplitudes, followed by automatic chain tracing methods, have allowed the rapid solution of large numbers of protein crystal structures. Despite extensive work, molecular replacement or the subsequent rebuilding usually fail with more divergent starting models based on remote homologues with less than 30% sequence identity. Here we show that this limitation can be substantially reduced by combining algorithms for protein structure modelling with those developed for crystallographic structure determination. An approach integrating Rosetta structure modelling with Autobuild chain tracing yielded high-resolution structures for 8 of 13 X-ray diffraction data sets that could not be solved in the laboratories of expert crystallographers and that remained unsolved after application of an extensive array of alternative approaches. We estimate that the new method should allow rapid structure determination without experimental phase information for over half the cases where current methods fail, given diffraction data sets of better than 3.2 Å resolution, four or fewer copies in the asymmetric unit, and the availability of structures of homologous proteins with >20% sequence identity.

EstB from Burkholderia gladioli: A novel esterase with a β‐lactamase fold reveals steric factors to discriminate between esterolytic and β‐lactam cleaving activity

Esterases form a diverse class of enzymes of largely unknown physiological role. Because many drugs and pesticides carry ester functions, the hydrolysis of such compounds forms at least one potential biological function. Carboxylesterases catalyze the hydrolysis of short chain aliphatic and aromatic carboxylic ester compounds. Esterases, d‐alanyl‐d‐alanine‐peptidases (DD‐peptidases) and β‐lactamases can be grouped into two distinct classes of hydrolases with different folds and topologically unrelated catalytic residues, the one class comprising of esterases, the other one of β‐lactamases and DD‐peptidases. The chemical reactivities of esters and β‐lactams towards hydrolysis are quite similar, which raises the question of which factors prevent esterases from displaying β‐lactamase activity and vice versa. Here we describe the crystal structure of EstB, an esterase isolated from Burkholderia gladioli. It shows the protein to belong to a novel class of esterases with homology to Penicillin binding proteins, notably DD‐peptidase and class C β‐lactamases. Site‐directed mutagenesis and the crystal structure of the complex with diisopropyl‐fluorophosphate suggest Ser75 within the “β‐lactamase” Ser‐x‐x‐Lys motif to act as catalytic nucleophile. Despite its structural homology to β‐lactamases, EstB shows no β‐lactamase activity. Although the nature and arrangement of active‐site residues is very similar between EstB and homologous β‐lactamases, there are considerable differences in the shape of the active site tunnel. Modeling studies suggest steric factors to account for the enzymes selectivity for ester hydrolysis versus β‐lactam cleavage.

Mechanism of cyanogenesis : the crystal structure of hydroxynitrile lyase from Hevea brasiliensis

BACKGROUND Over three thousand species of plants, including important food crops such as cassava, use cyanogenesis, the liberation of HCN upon tissue damage, as a defense against predation. Detoxification of cyanogenic food crops requires disruption of the cyanogenic pathway. Hydroxynitrile lyase is one of the key enzymes in cyanogenesis, catalyzing the decomposition of an alpha-cyanohydrin to form HCN plus the corresponding aldehyde or ketone. These enzymes are also of potential utility for industrial syntheses of optically pure chiral cyanohydrins, being used to catalyze the reverse reaction. We set out to gain insight into the catalytic mechanism of this important class of enzymes by determining the three-dimensional structure of hydroxynitrile lyase from the rubber tree, Hevea brasiliensis. RESULTS The crystal structure of the enzyme has been determined to 1.9 A resolution. It belongs to the alpha/beta hydrolase superfamily, with an active site that is deeply buried within the protein and connected to the outside by a narrow tunnel. The catalytic triad is made up of Ser80, His235 and Asp207. By analogy with known mechanisms of other members of this superfamily, catalysis should involve an oxyanion hole formed by the main chain NH of Cys81 and the side chains of Cys81 and Thr11. Density attributed to a histidine molecule or ion is found in the active site. CONCLUSIONS By analogy with other alpha/beta hydrolases, the reaction catalyzed by hydroxynitrile lyase involves a tetrahedral hemiketal or hemiacetal intermediate formed by nucleophilic attack of Ser80 on the substrate, stabilized by the oxyanion hole. The SH group of Cys81 is probably involved in proton transfer between the HCN and the hydroxynitrile OH. This mechanism is significantly different from the corresponding uncatalyzed solution reaction.

Enantiocomplementary enzymes: classification, molecular basis for their enantiopreference, and prospects for mirror-image biotransformations.

One often-cited weakness of biocatalysis is the lack of mirror-image enzymes for the formation of either enantiomer of a product in asymmetric synthesis. Enantiocomplementary enzymes exist as the solution to this problem in nature. These enzyme pairs, which catalyze the same reaction but favor opposite enantiomers, are not mirror-image molecules; however, they contain active sites that are functionally mirror images of one another. To create mirror-image active sites, nature can change the location of the binding site and/or the location of key catalytic groups. In this Minireview, X-ray crystal structures of enantiocomplementary enzymes are surveyed and classified into four groups according to how the mirror-image active sites are formed.

Atomic Resolution Crystal Structure of Hydroxynitrile Lyase from Hevea Brasiliensis

Abstract The X-ray crystal structure of native hydroxynitrile lyase from Hevea brasiliensis (Hb-HNL) has been determined at 1.1 Å resolution. It refined to a final R of 11.5% for all data and an Rfree of 14.4%. The favorable data-to-parameter ratio at atomic resolution made the refinement of individual anisotropic displacement parameters possible. The data also allowed a clear distinction of the alternate orientations of all histidine and the majority of asparagine and glutamine side chains. A number of hydrogen atoms, including one on the imidazole of the mechanistically important His-235, became visible as peaks in a difference electron density map. The structure revealed a discretely disordered sidechain of Ser-80, which is part of the putative catalytic triad. Analysis of the anisotropy indicated an increased mobility of residues near the entrance to the active site and within the active site.

Tautomerism and stereochemistry of hypericin: Force field, NMR, and X-ray crystallographic investigations

SummaryStereochemistry and tautomerism of hypericin, pseudohypericin, and several of their partial structure models were investigated using an MM2 derived force field method. Besides the “propeller” type conformer, which was also found by the X-ray crystallographic study, the complicated energy hypersurface was shown to contain a novel “double-butterfly” conformer of similar stability. The upper limit interconversion barrier between these conformers and their enantiomers was found to be in the order of 115 kJ/mol.1H-NMR experiments suggested a lower limit interconversion barrier of at least 80 kJ/mol. From the ten tautomers possible in principle, the 7,14-species was derived to be the most stable one by at least 48 kJ/mol.ZusammenfassungDie Stereochemie von Hypericin, Pseudohypericin und einigen seiner Partialstrukturmodelle wurde mit Hilfe einer von MM2 abgeleiteten Kraftfeldmethodik untersucht. In der komplizierten Energiehyperfläche wurde neben dem auch durch Röntgenstrukturanalyse gefundenen „Propeller“-Konformeren ein neues „Doppelschmetterling“-Konformer ähnlicher Stabilität aufgefunden. Die obere Grenze für die Interkonversionsbarrieren zwischen diesen Konformeren und ihren Enantiomeren sind in der Größenordnung von 115 kJ/mol. Aus1H-NMR-Experimenten konnte eine untere Grenze von wenigstens 80 kJ/mol abgeleitet werden. Es wurde gefunden, daß von den zehn prinzipiell möglichen Tautomeren die 7,14-Spezies die um wenigstens 48 kJ/mol stabilste ist.

Structure of the molybdate/tungstate binding protein mop from Sporomusa ovata.

BACKGROUND Transport of molybdenum into bacteria involves a high-affinity ABC transporter system whose expression is controlled by a repressor protein called ModE. While molybdate transport is tightly coupled to utilization in some bacteria, other organisms have molybdenum storage proteins. One class of putative molybdate storage proteins is characterized by a sequence consisting of about 70 amino acids (Mop). A tandem repeat of Mop sequences also constitutes the molybdate binding domain of ModE. RESULTS We have determined the crystal structure of the 7 kDa Mop protein from the methanol-utilizing anaerobic eubacterium Sporomusa ovata grown in the presence of molybdate and tungstate. The protein occurs as highly symmetric hexamers binding eight oxyanions. Each peptide assumes a so-called OB fold, which has previously also been observed in ModE. There are two types of oxyanion binding sites in Mo at the interface between two or three peptides. All oxyanion binding sites were found to be occupied by WO(4) rather than MoO(4). CONCLUSIONS The biological function of proteins containing only Mop sequences is unknown, but they have been implicated in molybdate homeostasis and molybdopterin cofactor biosynthesis. While there are few indications that the S. ovata Mop binds pterin, the structure suggests that only the type-1 oxyanion binding sites would be sufficiently accessible to bind a cofactor. The observed occupation of the oxyanion binding sites by WO(4) indicates that Mop might also be involved in controlling intracellular tungstate levels.

Towards automated Laue data processing: application to the choice of optimal X-ray spectrum

Laue data reduction has now reached a level of sophistication that allows nearly automated processing to be performed. The software described enables complete reduction of the data with essentially no user intervention, making Laue processing almost as straightforward as monochromatic data processing. Interactive work is limited to the indexing of only one Laue pattern. More importantly, it is shown that the data quality is substantially enhanced when soft-limited predictions are used. Further improvement obtained by taking advantage of the structure-factor amplitudes from a known closely related structure is described. To determine the most suitable type of insertion device to be used for time-resolved Laue crystallography, the technique described was applied to Laue data sets collected from photoactive yellow protein under identical conditions but with three different insertion devices: a wiggler, a broad-bandpass undulator and a single-line undulator. Although the optimal choice may ultimately be dictated by sample parameters (such as mosaic spread) and by the type of experiment (repeatable or non-repeatable reactions), the results here show that the use of single-line undulators will generally yield by far the best compromise between data quality, acquisition time and radiation damage.

Beiträge zur Chemie der Pyrrolpigmente, 77. Mitt. Synthese und Struktur vonb-Nor-bilatrienen-abc undb-Nor-biladienen-ac bzw. Bi-9,9′-dipyrrinonylidenen und Bi-9,9′-dipyrrinonylenen

B-Nor-biladienes-ac are prepared by oxidative coupling of dipyrrinones in nearly quantitative yields. Further oxidation of these derivatives yieldsb-nor-bilatrienes-abc. Constitutions, tautomerism, configurations at exocyclic double bonds, and conformations at exocyclic chromophore single bonds are determined for the crystalline state and their states of solution using X-ray structural analysis and spectroscopic methods, respectively.B-nor-biladienes-ac are found to be of (4Z,15Z)5sp,9sc,14sp geometry,b-nor-bilatrienes-abc are planar systems of (4Z,9E,15Z) configuration in the crystal, whereas in solutions there are indications of a more twisted conformation at their exocyclic chromophore single bonds.

A Stereoselective Inverting sec-Alkylsulfatase for the Deracemization of sec-Alcohols

A metallo-β-lactamase-type alkylsulfatase was found to catalyze the enantioselective hydrolysis of sec-alkylsulfates with strict inversion of configuration. This catalytic event, which does not have an analog in chemocatalysis, yields homochiral (S)-configurated alcohols and nonreacted sulfate esters. The latter could be converted into (S)-sec-alcohols as the sole product in up to >99% ee via a chemoenzymatic deracemization protocol on a preparative scale.