Multiple regulatory mechanisms control the expression of the Geobacillus stearothermophilus gene for extracellular xylanase.

Geobacillus stearothermophilus T-6 produces a single extracellular xylanase (Xyn10A) capable of producing short, decorated xylo-oligosaccharides from the naturally branched polysaccharide, xylan. Gel retardation assays indicated that the master negative regulator, XylR, binds specifically to xylR operators in the promoters of xylose and xylan-utilization genes. This binding is efficiently prevented in vitro by xylose, the most likely molecular inducer. Expression of the extracellular xylanase is repressed in medium containing either glucose or casamino acids, suggesting that carbon catabolite repression plays a role in regulating xynA. The global transcriptional regulator CodY was shown to bind specifically to the xynA promoter region in vitro, suggesting that CodY is a repressor of xynA. The xynA gene is located next to an uncharacterized gene, xynX, that has similarity to the NIF3 (Ngg1p interacting factor 3)-like protein family. XynX binds specifically to a 72-bp fragment in the promoter region of xynA, and the expression of xynA in a xynX null mutant appeared to be higher, indicating that XynX regulates xynA. The specific activity of the extracellular xylanase increases over 50-fold during early exponential growth, suggesting cell density regulation (quorum sensing). Addition of conditioned medium to fresh and low cell density cultures resulted in high expression of xynA, indicating that a diffusible extracellular xynA density factor is present in the medium. The xynA density factor is heat-stable, sensitive to proteases, and was partially purified using reverse phase liquid chromatography. Taken together, these results suggest that xynA is regulated by quorum-sensing at low cell densities.

Carbohydrate-Binding Capability and Functional Conformational Changes of AbnE, an Arabino-oligosaccharide Binding Protein.

ABC importers are membrane proteins responsible for the transport of nutrients into the cells of prokaryotes. Although the structures of ABC importers vary, all contain four conserved domains: two nucleotide-binding domains (NBDs), which bind and hydrolyze ATP, and two transmembrane domains (TMDs), which help translocate the substrate. ABC importers are also dependent on an additional protein component, a high-affinity substrate-binding protein (SBP) that specifically binds the target ligand for delivery to the appropriate ABC transporter. AbnE is a SBP belonging to the ABC importer for arabino-oligosaccharides in the Gram-positive thermophilic bacterium Geobacillus stearothermophilus. Using isothermal titration calorimetry (ITC), purified AbnE was shown to bind medium-sized arabino-oligosaccharides, in the range of arabino-triose (A3) to arabino-octaose (A8), all with Kd values in the nanomolar range. We describe herein the 3D structure of AbnE in its closed conformation in complex with a wide range of arabino-oligosaccharide substrates (A2-A8). These structures provide the basis for the detailed structural analysis of the AbnE-sugar complexes, and together with complementary quantum chemical calculations, site-specific mutagenesis, and isothermal titration calorimetry (ITC) experiments, provide detailed insights into the AbnE-substrate interactions involved. Small-angle X-ray scattering (SAXS) experiments and normal mode analysis (NMA) are used to study the conformational changes of AbnE, and these data, taken together, suggest clues regarding its binding mode to the full ABC importer.

Enhancement of COPD biological networks using a web-based collaboration interface.

The construction and application of biological network models is an approach that offers a holistic way to understand biological processes involved in disease. Chronic obstructive pulmonary disease (COPD) is a progressive inflammatory disease of the airways for which therapeutic options currently are limited after diagnosis, even in its earliest stage. COPD network models are important tools to better understand the biological components and processes underlying initial disease development. With the increasing amounts of literature that are now available, crowdsourcing approaches offer new forms of collaboration for researchers to review biological findings, which can be applied to the construction and verification of complex biological networks. We report the construction of 50 biological network models relevant to lung biology and early COPD using an integrative systems biology and collaborative crowd-verification approach. By combining traditional literature curation with a data-driven approach that predicts molecular activities from transcriptomics data, we constructed an initial COPD network model set based on a previously published non-diseased lung-relevant model set. The crowd was given the opportunity to enhance and refine the networks on a website ( https://bionet.sbvimprover.com/) and to add mechanistic detail, as well as critically review existing evidence and evidence added by other users, so as to enhance the accuracy of the biological representation of the processes captured in the networks. Finally, scientists and experts in the field discussed and refined the networks during an in-person jamboree meeting. Here, we describe examples of the changes made to three of these networks: Neutrophil Signaling, Macrophage Signaling, and Th1-Th2 Signaling. We describe an innovative approach to biological network construction that combines literature and data mining and a crowdsourcing approach to generate a comprehensive set of COPD-relevant models that can be used to help understand the mechanisms related to lung pathobiology. Registered users of the website can freely browse and download the networks.

Aroma biosynthesis in strawberry: s-adenosylmethionine:furaneol o-methyltransferase activity in ripening fruits.

Among the most important volatile compounds in the aroma of strawberries are 2,5-dimethyl-4-hydroxy-3(2H)-furanone (Furaneol) and its methoxy derivative (methoxyfuraneol, mesifuran). Three strawberry varieties, Malach, Tamar, and Yael, were assessed for total volatiles, Furaneol, and methoxyfuraneol. The content of these compounds sharply increased during fruit ripening, with maximum values at the ripe stage. An enzymatic activity that transfers a methyl group from S-adenosylmethionine (SAM) to Furaneol sharply increases during ripening of strawberry fruits. The in vitro generated methoxyfuraneol was identified by radio-TLC and GC-MS. The partially purified enzyme had a native molecular mass of approximately 80 kDa, with optimum activity at pH 8.5 and 37 degrees C. A high apparent K(m) of 5 mM was calculated for Furaneol, whereas this enzyme preparation apparently accepted as substrates other o-dihydroxyphenol derivatives (such as catechol, caffeic acid, and protocatechuic aldehyde) with much higher affinities (K(m) approximately 105, 130, and 20 microM, respectively). A K(m) for SAM was found to be approximately 5 microM, regardless of the acceptor used. Substrates that contained a phenolic group with only one OH group, such as p-coumaric and trans-ferulic acid, as well as trans-anol and coniferyl alcohol, were apparently not accepted by this activity. It is suggested that Furaneol methylation is mediated by an O-methyltransferase activity and that this activity increases during fruit ripening.

Preliminary crystallographic analysis of Xyn52B2, a GH52 ß-D-xylosidase from Geobacillus stearothermophilus T6.

Geobacillus stearothermophilus T6 is a thermophilic bacterium that possesses an extensive hemicellulolytic system, including over 40 specific genes that are dedicated to this purpose. For the utilization of xylan, the bacterium uses an extracellular xylanase which degrades xylan to decorated xylo-oligomers that are imported into the cell. These oligomers are hydrolyzed by side-chain-cleaving enzymes such as arabinofuranosidases, acetylesterases and a glucuronidase, and finally by an intracellular xylanase and a number of ß-xylosidases. One of these ß-xylosidases is Xyn52B2, a GH52 enzyme that has already proved to be useful for various glycosynthesis applications. In addition to its demonstrated glycosynthase properties, interest in the structural aspects of Xyn52B2 stems from its special glycoside hydrolase family, GH52, the structures and mechanisms of which are only starting to be resolved. Here, the cloning, overexpression, purification and crystallization of Xyn52B2 are reported. The most suitable crystal form that has been obtained belonged to the orthorhombic P212121 space group, with average unit-cell parameters a = 97.7, b = 119.1, c = 242.3 Å. Several X-ray diffraction data sets have been collected from flash-cooled crystals of this form, including the wild-type enzyme (3.70 Šresolution), the E335G catalytic mutant (2.95 Šresolution), a potential mercury derivative (2.15 Šresolution) and a selenomethionine derivative (3.90 Šresolution). These data are currently being used for detailed three-dimensional structure determination of the Xyn52B2 protein.

Two active forms of Zymomonas mobilis levansucrase. An ordered microfibril structure of the enzyme promotes levan polymerization.

Fructansucrases, members of glycoside hydrolase family 68, catalyze both sucrose hydrolysis and the polymerization of fructose to beta-d-fructofuranose polymers. The resulting fructan polymers are distinguished by the nature of the glycosidic bond: inulin (beta-(2-1)-fructofuranose) and levan (beta-(2-6)-fructofuranose). In this study we demonstrate that Zymomonas mobilis levansucrase exists in two active forms, depending on the pH and ionic strength. At pH values above 7.0, the enzyme is mainly a dimer, whereas at pH values below 6.0, the protein forms well ordered microfibrils that precipitate out of the solution. These two forms are readily interchangeable simply by changing the pH. Surprisingly the manner in which the enzyme is arranged strongly affects its product specificity and kinetic properties. At pH values above 7.0, the activity of the enzyme as a dimer is mainly sucrose hydrolysis and the synthesis of short fructosaccharides (degree of polymerization, 3). At pH values below 6.0, in its microfibril form, the enzyme catalyzes almost exclusively the synthesis of levan (a degree of polymerization greater than 20,000). This difference in product specificity appears to depend on the form of the enzyme, dimer versus microfibril, and not directly on the pH. Images made by negative stain transmission electron microscopy reveal that the enzyme forms a very ordered structure of long fibrils that appear to be composed of repeating rings of six to eight protein units. A single amino acid replacement of H296R abolished the ability of the enzyme to form microfibrils with organized fibril networks and to synthesize levan at pH 6.0.

A programmable biomolecular computing machine with bacterial phenotype output.

The main advantage of autonomous biomolecular computing devices over electronic computers is their ability to interact directly with biological systems. No interface is required since all components of molecular computers, including hardware, software, input, and output are molecules that interact in solution along a cascade of programmable chemical events. Here, we demonstrate for the first time that the output of a computation preduced by a molecular finite automaton can be a visible bacterial phenotype. Our 2-symbol-2-state finite automaton utilized linear double-stranded DNA inputs that were prepared by inserting a string of six base pair symbols into the lacZ gene on the pUC18 plasmid. The computation resulted in a circular plasmid that differed from the original pUC18 by either a 9 base pair (accepting state) or 11 base pair insert (unaccepting state) within the lacZ alpha region gene. Upon transformation and expression of the resultant plasmids in E. coli, the accepting state was represented by production of functional beta-galactosidase and formation of blue colonies on X-gal medium. In contrast, the unaccepting state was represented by white colonies due to a shift in the open reading frame of lacZ.

Isolation, cloning and expression of a multifunctional O-methyltransferase capable of forming 2,5-dimethyl-4-methoxy-3(2H)-furanone, one of the key aroma compounds in strawberry fruits.

Strawberry fruits contain an uncommon group of key aroma compounds with a 2,5-dimethyl-3(2H)-furanone structure. Here, we report on the methylation of 2,5-dimethyl-4-hydroxy-3(2H)-furanone (DMHF) to 2,5-dimethyl-4-methoxy-3(2H)-furanone (DMMF) by a S-adenosyl-L-methionine dependent O-methyltransferase, the cloning of the corresponding cDNA and characterization of the encoded protein. Northern-hybridization indicated that the Strawberry-OMT specific transcripts accumulated during ripening in strawberry fruits and were absent in root, petiole, leaf and flower. The protein was functionally expressed in E. coli and exhibited a substrate specificity for catechol, caffeic acid, protocatechuic aldehyde, caffeoyl CoA and DMHF. A common structural feature of the accepted substrates was a o-diphenolic structure also present in DMHF in its dienolic tautomer. FaOMT is active as a homodimer and the native enzyme shows optimum activity at pH 8.5 and 37 degrees C. It does not require a cofactor for enzymatic activity. Due to the expression pattern of FaOMT and the enzymatic activity in the different stages of fruit ripening we suppose that FaOMT is involved in lignification of the achenes and the vascular bundles in the expanding fruit. In addition, it is concluded that the Strawberry-OMT plays an important role in the biosynthesis of strawberry volatiles such as vanillin and DMMF.

Characterization of phenylpropene O-methyltransferases from sweet basil: facile change of substrate specificity and convergent evolution within a plant O-methyltransferase family.

Some basil varieties are able to convert the phenylpropenes chavicol and eugenol to methylchavicol and methyleugenol, respectively. Chavicol O-methyltransferase (CVOMT) and eugenol O-methyltransferase (EOMT) cDNAs were isolated from the sweet basil variety EMX-1 using a biochemical genomics approach. These cDNAs encode proteins that are 90% identical to each other and very similar to several isoflavone O-methyltransferases such as IOMT, which catalyzes the 4'-O-methylation of 2,7,4'-trihydroxyisoflavanone. On the other hand, CVOMT1 and EOMT1 are related only distantly to (iso)eugenol OMT from Clarkia breweri, indicating that the eugenol O-methylating enzymes in basil and C. breweri evolved independently. Transcripts for CVOMT1 and EOMT1 were highly expressed in the peltate glandular trichomes on the surface of the young basil leaves. The CVOMT1 and EOMT1 cDNAs were expressed in Escherichia coli, and active proteins were produced. CVOMT1 catalyzed the O-methylation of chavicol, and EOMT1 also catalyzed the O-methylation of chavicol with equal efficiency to that of CVOMT1, but it was much more efficient in O-methylating eugenol. Molecular modeling, based on the crystal structure of IOMT, suggested that a single amino acid difference was responsible for the difference in substrate discrimination between CVOMT1 and EOMT1. This prediction was confirmed by site-directed mutagenesis, in which the appropriate mutants of CVOMT1 (F260S) and EOMT1 (S261F) were produced that exhibited the opposite substrate preference relative to the respective native enzyme.

O-methyltransferases involved in the biosynthesis of volatile phenolic derivatives in rose petals.

Rose (Rosa hybrida) flowers produce and emit a diverse array of volatiles, characteristic to their unique scent. One of the most prominent compounds in the floral volatiles of many rose varieties is the methoxylated phenolic derivative 3,5-dimethoxytoluene (orcinol dimethyl ether). Cell-free extracts derived from developing rose petals displayed O-methyltransferase (OMT) activities toward several phenolic substrates, including 3,5-dihydroxytoluene (orcinol), 3-methoxy,5-hydroxytoluene (orcinol monomethyl ether), 1-methoxy, 2-hydroxy benezene (guaiacol), and eugenol. The activity was most prominent in rose cv Golden Gate, a variety that produces relatively high levels of orcinol dimethyl ether, as compared with rose cv Fragrant Cloud, an otherwise scented variety but which emits almost no orcinol dimethyl ether. Using a functional genomics approach, we have identified and characterized two closely related cDNAs from a rose petal library that each encode a protein capable of methylating the penultimate and immediate precursors (orcinol and orcinol monomethyl ether, respectively) to give the final orcinol dimethyl ether product. The enzymes, designated orcinol OMTs (OOMT1 and OOMT2), are closely related to other plant methyltransferases whose substrates range from isoflavones to phenylpropenes. The peak in the levels of OOMT1 and OOMT2 transcripts in the flowers coincides with peak OMT activity and with the emission of orcinol dimethyl ether.