Sari Paavilainen

Structural analysis, enzymatic characterization, and catalytic mechanisms of β‐galactosidase from Bacillus circulans sp. alkalophilus

Crystal structures of native and α‐d‐galactose‐bound Bacillus circulans sp. alkalophilusβ‐galactosidase (Bca‐β‐gal) were determined at 2.40 and 2.25 Å resolutions, respectively. Bca‐β‐gal is a member of family 42 of glycoside hydrolases, and forms a 460 kDa hexameric structure in crystal. The protein consists of three domains, of which the catalytic domain has an (α/β)8 barrel structure with a cluster of sulfur‐rich residues inside the β‐barrel. The shape of the active site is clearly more open compared to the only homologous structure available in the Protein Data Bank. This is due to the number of large differences in the loops that connect the C‐terminal ends of the β‐strands to the N‐terminal ends of the α‐helices within the (α/β)8 barrel. The complex structure shows that galactose binds to the active site as an α‐anomer and induces clear conformational changes in the active site. The implications of α‐d‐galactose binding with respect to the catalytic mechanism are discussed. In addition, we suggest that β‐galactosidases mainly utilize a reverse hydrolysis mechanism for synthesis of galacto‐oligosaccharides.

Crystal structure of the ligand-binding domain of the promiscuous EphA4 receptor reveals two distinct conformations.

Eph receptors and their ephrin ligands are important mediators of cell-cell communication. They are divided in two subclasses based on their affinities for each other and on sequence conservation. Receptor-ligand binding within each subclass is fairly promiscuous, while binding cross the subclasses happens rarely. EphA4 is an exception to this general rule, since it has long been known to bind both A- and B-class ephrin ligands but the reason for this exceptional behavior has not been worked out at molecular level. Recent structural and biochemical studies on EphA4 ligand-binding domain alone and in complex with its ligands have addressed this question. However, the published structures of EphA4/ephrin complexes differ considerably from each other and strikingly different explanations for the exceptional promiscuity of EphA4 were proposed. To address these contradictory findings, we have determined a crystal structure of the EphA4 ligand-binding domain at 2.3A resolution and show that the receptor has an unprecedented ability to exist in two very different, well-ordered conformations even in the unbound state. Our results suggest that the ligand promiscuity of the Ephs is directly correlated with the structural flexibility of the ligand-binding surface of the receptor.

Conversion of Carbohydrates to Organic Acids by Alkaliphilic Bacilli

Abstract Sugar catabolism of alkaliphilic bacilli was investigated to reveal their catabolic characteristics. Four bacilli were cultured in the presence of various 1% (w/v) sugars and related compounds such as sugar alcohols, and the catabolites formed were analyzed. All alkaliphiles produced acetic acid (4.5–5 g/l at the maximum) while formic acid was produced by only one of the strains. In contrast to neutrophilic bacilli, acetoin, butanediol or ethanol was not detected. Moderate amounts of isobutyric, isovaleric, α-oxoisovaleric, α-oxo-β-methylvaleric, α-oxoisocaproic and phenylacetic acids were generated with three of the alkaliphiles. High pH and/or buffer concentration tends to favour formation of acids. The studied alkaliphiles are compared among each other and with neutrophilic bacilli.

Comparison of high-performance liquid and gas chromatography in the determination of organic acids in culture media of alkaliphilic bacteria

Abstract Volatile and non-volatile organic acids were analysed by high-performance liquid chromatography (HPLC) on an Aminex HPX-87H, gas-solid chromatography (GSC) on a Porapak Q and gas-liquid chromatography (GLC) on a fused-silica capillary column. The results were compared by using standard acid solutions and with culture media of two strains of alkaliphilic Bacillus. Whereas the resolution of acids was excellent with GLC, the quantitative reproducibility was better with HPLC. Identification of complex culture mixtures was accomplished by GC-MS. Applicability of the methods for different purposes is discussed.

Enhanced conversion of starch to cyclodextrins in ethanolic solutions by Bacillus circulans var alkalophilus cyclomaltodextrin glucanotransferase

Improved formation of cyclodextrins (CDs) from starch in ethanolic solutions byBacillus circulans var alkalophilus cyclomaltodextrin glucanotransferase was studied. The β- and γ-CD yields increased and α-CD yield gradually decreased as the ethanol concentration was raised. The ethanol concentration required for maximal CD yield depended essentially on starch concentration. The ethanols effect was pronounced at high starch concentrations. For example, with 30% (w/v) starch, the CD yield was 2.4-fold (146.5 g/L) in the presence of 15% (v/v) ethanol. The effect of dimethylsulfoxide on the formation of CDs was similar to that of ethanol. The disintegration of β- and γ-CDs were narrowly interdependent on the formation of a α-CD and malto-sugars. The amount of reducing sugars decreased from a dextrose equivalent value of roughly 7.5 to 4.5 in the presence of ethanol at starch concentrations 1-30% (w/v). The effect of ethanol on starchy materials from various sources was similar. It was concluded that ethanol retards the decomposition of β-CD by a general mechanism involving a decreased activity of water.

Characterization of bacterial growth on solid medium with image analysis.

Alkaliphilic bacterial strains producing the enzyme cyclodextrin glucanotransferase were cultivated on solid agar medium containing an indicator system detecting the enzyme. The growth of the colony and the surrounding diffusion zone, due to the enzyme, were measured by the image analysis during the cultivation. It was possible to differentiate between relatively similar clones by observing quantitatively the changes at and around the colony. Optimal experimental conditions for such measurements are discussed. The image analysis technique provides a potential tool for characterizing microbes grown on solid media.

Structural basis for Myf and Psa fimbriae-mediated tropism of pathogenic strains of Yersinia for host tissues.

Three pathogenic species of the genus Yersinia assemble adhesive fimbriae via the FGL‐chaperone/usher pathway. Closely related Y. pestis and Y. pseudotuberculosis elaborate the pH6 antigen (Psa), which mediates bacterial attachment to alveolar cells of the lung. Y. enterocolitica, instead, assembles the homologous fimbriae Myf of unknown function. Here, we discovered that Myf, like Psa, specifically recognizes β1‐3– or β1‐4–linked galactose in glycosphingolipids, but completely lacks affinity for phosphatidylcholine, the main receptor for Psa in alveolar cells. The crystal structure of a subunit of Psa (PsaA) complexed with choline together with mutagenesis experiments revealed that PsaA has four phosphatidylcholine binding pockets that enable super‐high‐avidity binding of Psa‐fibres to cell membranes. The pockets are arranged as six tyrosine residues, which are all missing in the MyfA subunit of Myf. Conversely, the crystal structure of the MyfA‐galactose complex revealed that the galactose‐binding site is more extended in MyfA, enabling tighter binding to lactosyl moieties. Our results suggest that during evolution, Psa has acquired a tyrosine‐rich surface that enables it to bind to phosphatidylcholine and mediate adhesion of Y. pestis/pseudotuberculosis to alveolar cells, whereas Myf has specialized as a carbohydrate‐binding adhesin, facilitating the attachment of Y. enterocolitica to intestinal cells.

Structural Insight into Archaic and Alternative Chaperone-Usher Pathways Reveals a Novel Mechanism of Pilus Biogenesis

Gram-negative pathogens express fibrous adhesive organelles that mediate targeting to sites of infection. The major class of these organelles is assembled via the classical, alternative and archaic chaperone-usher pathways. Although non-classical systems share a wider phylogenetic distribution and are associated with a range of diseases, little is known about their assembly mechanisms. Here we report atomic-resolution insight into the structure and biogenesis of Acinetobacter baumannii Csu and Escherichia coli ECP biofilm-mediating pili. We show that the two non-classical systems are structurally related, but their assembly mechanism is strikingly different from the classical assembly pathway. Non-classical chaperones, unlike their classical counterparts, maintain subunits in a substantially disordered conformational state, akin to a molten globule. This is achieved by a unique binding mechanism involving the register-shifted donor strand complementation and a different subunit carboxylate anchor. The subunit lacks the classical pre-folded initiation site for donor strand exchange, suggesting that recognition of its exposed hydrophobic core starts the assembly process and provides fresh inspiration for the design of inhibitors targeting chaperone-usher systems.

Structure of the ligand-binding domain of the EphB2 receptor at 2 A resolution.

Eph tyrosine kinase receptors, the largest group of receptor tyrosine kinases, and their ephrin ligands are important mediators of cell-cell communication regulating cell attachment, shape and mobility. Recently, several Eph receptors and ephrins have also been found to play important roles in the progression of cancer. Structural and biophysical studies have established detailed information on the binding and recognition of Eph receptors and ephrins. The initial high-affinity binding of Eph receptors to ephrin occurs through the penetration of an extended G-H loop of the ligand into a hydrophobic channel on the surface of the receptor. Consequently, the G-H loop-binding channel of Eph receptors is the main target in the search for Eph antagonists that could be used in the development of anticancer drugs and several peptides have been shown to specifically bind Eph receptors and compete with the cognate ephrin ligands. However, the molecular details of the conformational changes upon Eph/ephrin binding have remained speculative, since two of the loops were unstructured in the original model of the free EphB2 structure and their conformational changes upon ligand binding could consequently not be analyzed in detail. In this study, the X-ray structure of unbound EphB2 is reported at a considerably higher 2 A resolution, the conformational changes that the important receptor loops undergo upon ligand binding are described and the consequences that these findings have for the development of Eph antagonists are discussed.

Proton and Carbon Inventory during the Growth of an Alkaliphilic Bacillus Indicates That Protons are Independent from Acid Anions

Abstract Sugar catabolism and pH-dynamics in culture medium of an alkaliphilic bacterium, Bacillus circulans var. alkalophilus were studied. The moles of carbon in 1% glucose (55 mmol/ l ) media were recovered almost quantitatively in acetic acid (65–75 mmol/ l ), formic acid (40–45 mmol/ l ) and CO 2 (90–100 mmol/ l ). The moles of acids needed to achieve the minimum pH of the medium during bacterial growth approximately corresponded to the moles of produced acids. However, the timing of the pH minimum did not match with the appearance of the acids, but the required amount of acids were detected several hours later in the medium. During active growth, the concentration of dissolved oxygen remained zero at all reasonable aeration levels in a fermentor. It was concluded that sugars are not metabolized through the tricarboxylic acid cycle, but the reduction potential generated from dehydrogenation of sugars is the driving force for the growth followed by the very effective transfer of hydrogen atoms/protons onto molecular oxygen/oxygen anion. The eventual rise of the pH shall result from re-equilibration of the redox state, and not from consumption of acids or generation of basic compounds as suggested previously.