Thursday, December 1, 2011

First observation of metamorphosis of an enzyme that catalyzes two chemical reactions

Archaea possess many unusual enzymes. Among them, fructose-1,6-bisphosphate (FBP) aldolase/phosphatase is especially peculiar. In apparent violation of biochemical canons, this catalyzes two fundamentally different chemical reactions. Additionally, this enzyme is responsible for the biosynthesis of glucose, a process of critical importance in the early stage of the .

The research team utilized the Photon Factory at KEK to observe the enzyme’s structural metamorphosis to bring about the catalysis of the two different reactions. Such a multifunctional enzyme shatters long-held beliefs in biochemistry and raises the intriguing possibility that more such enzymes might be discovered in other organisms.

Hyperthermophiles live in ultra-hot water and are positioned near the root of the evolutionary tree of life. As such, they are thought to be close to the common ancestor of life on Earth. Many hyperthermophiles are categorized as archaea, which belong to different branches from those of common organisms, such as bacteria and eukaryotes. Some hyperthermophilic archaea have the ability to build their own bodies by synthesizing complex compounds from simple inorganic substances, for example in the biosynthesis of saccharides from carbon dioxide. Reaction pathways in which organisms synthesize glucose are thought to be important in the evolution of primitive organisms.

First observation of metamorphosis of an enzyme that catalyzes two chemical reactions

Figure 3. Reactions that are catalyzed by FBPA/P (left) and schematic views of regions where reactions occur (right). Three loops undergo significant conformational changes in changing between the FBP aldolase form (top right) and the FBP phosphatase form (bottom right).

For most common organisms two different enzymes, fructose-1,6-bisphosphate (FBP) aldolase and FBP phosphatase, are sequentially responsible for chemical reactions that lead to production. By contrast, hyperthermophilic archaea make use of a single protein, termed FBP aldolase/phosphatase (FBPA/P), to catalyze these two reactions. This enzyme could be said to be bifunctional.

The research team studied FBPA/P found in the hyperthermophilic archaeon, Sulfolobus, isolated from hot water at Beppu Onsen in Oita Prefecture, Japan. The three-dimensional structure of FBPA/P looks similar to a barrel of eight identical molecular units. Reactions are catalyzed in the regions between these units.

The two reactions that FBPA/P catalyzes are: 1) FBP aldolase reaction, in which dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (GA3P) produce FBP and 2) FBP phosphatase reaction, in which FBP is cleaved into fructose-6-phosphate (F6P) and inorganic phosphate (Pi).

In 2004, the research team elucidated the three-dimensional structure of the enzyme-substrate complex while FBPA/P is catalyzing the FBP phosphatase reaction. In the present study, the same group succeeded in unraveling the three-dimensional form of the enzyme-substrate complex while FBPA/P is catalyzing the FBP aldolase reaction by using X-ray crystallography in their experiments performed at the AR-NW12A beamline of the KEK’s Photon Factory. Comparison of these two forms revealed that the active site (the region of an enzyme in which a chemical reaction is catalyzed) of FBPA/P metamorphoses into two completely different forms owing to the extensive movements of three loops (lid loop, Schiff-base loop, and C-terminal loop).

In the FBP aldolase form, a lysine residue (K232) binds to the first substrate, DHAP. When the second substrate, GA3P, enters the active center, a neighboring tyrosine residue (Y229) is thought to mediate reaction of GA3P and DHAP into FBP (FBP aldolase reaction). Once FBP is produced, the lysine residue becomes free and the three loops can move. The Schiff-base loop is flipped over and both the lid and C-terminal loops close, metamorphosing the enzyme into its FBP phosphatase form. Next, an aspartate residue (D233) enters the active center, and binds magnesium ions, thereby triggering the dissociation of FBP into F6P and Pi (FBP reaction).

This finding revealed that such a ‘metamorphosis’ is the key to the catalysis of two different reactions for a given enzymatic reaction sequence. This mechanism turns on its head the established biochemical dogma that describes one enzyme as catalyzing only one reaction.

Common organisms of more recent ancestry do not possess FBPA/P, but rather make use of two different enzymes separately to catalyze the two reactions independently. It is possible that primitive organisms might perform in a simpler manner than more contemporary organisms do by utilizing bifunctional enzymes such as FBPA/P.

The present study is the first to reveal the mechanism by which one enzyme may catalyze two different . Moreover, the current finding hints at the possibility that more multifunctional enzymes similar to FBPA/P may exist. In addition, it can be expected that more enzymes capable of converting simple starting materials into synthetically useful intermediates will be discovered.

More information: Shinya Fushinobu, et al. “Structural basis for the bifunctionality of fructose 1,6-bisphosphate aldolase/phosphatase.” Nature, Online Edition. October 10, 2011. DOI:10.1038/nature10457

Provided by KEK

Bidentate chelates with larger spacers: Chelating Lewis acids prepared by double hydroalumination of dialkynylsilanes

Chelating Lewis acids with a geminal arrangement of two acceptor functions have been shown to coordinate halide, thiolate, or benzoate . The remarkable efficacy of the chelating coordination of hydride ions by two aluminum atoms by the formation of persistent carbocations through C-H bond activation is also known. However, often the acceptor atoms occupy geminal positions at a bridging carbon atom, which results in relatively strained four-membered heterocycles upon coordination of single-atom donors.

Therefore, a team of scientists led by Werner Uhl of the University of Münster (Germany) were very much interested in synthesizing that have larger spacers between the acceptor functions in order to obtain more flexible backbones and hence better coordinating properties. The twofold hydroalumination of silicon-centered dialkynes was employed as a facile route for the preparation of such compounds, as reported in the European Journal of Inorganic Chemistry.

During the double hydroalumination of dialkynylsilanes, mixed alkenyl–alkynyl compounds resulting from the reduction of only one C?C triple bond were obtained as intermediates, two of which were isolated and characterized. Hydroalumination of the remaining C?C triple bond yielded dialkenyl species that were ideally preorganized to be applied as chelating Lewis acids, which was demonstrated by the chelation of chloride ions. In addition, an alkenyl–alkynylsilane intermediate gave a silacyclobutene derivative by 1,1-carbalumination; this is the second time such a reaction has been observed. The mechanism of this reaction was investigated by quantum chemical calculations.

This study reports an easy way to synthesize chelating Lewis acids with two geminal acceptor aluminum atoms. The chelating coordination of chloride ions by both aluminum atoms to give a six-membered ClAl2C2Si heterocycle was demonstrated.

More information: Werner Uhl, Hydroalumination of Bis(alkynyl)silanes: Generation of Chelating Lewis Acids, Their Application in the Coordination of Chloride Ions and a 1,1-Carbalumination Reaction, European Journal of Inorganic Chemistry, … ic.201100890

Provided by Wiley (news : web)

Getting to xenon: Scientists examine alternatives for pulling this rare, expensive element out of air samples

"The promise of NiDOBDC and similar metal-organic frameworks is that we will be able to make faster and more sensitive separation and ," said Grate, who has been working in materials and systems for sensing and nonproliferation applications for his entire career at PNNL.

Absorbents, like activated charcoal and MOFs, can selectively capture and release it on demand. Making these absorbents more efficient could benefit sustainable processes and national security. Commercial uses of xenon include lighting, scientific instruments, and anesthesia. Security uses focus on nuclear processes. Nuclear reprocessing, weapons tests, and nuclear accidents, such as the 2011 catastrophe in Japan, release xenon into the atmosphere. Around the world, monitors track xenon for the Comprehensive Test Ban Treaty.

The research team compared the performance of three materials that capture xenon. Conventional technologies for capturing xenon use activated charcoal, which is fine powder processed to be porous, with an effective surface area of 500 square meters a gram. Charcoal also absorbs xenon from and releases it on demand.

"Activated charcoal is a really old-fashioned material with little potential for improvement," said Grate. "We wanted to see if we could find a material that we could push, that we could get new capabilities from."

Metal-organic frameworks or MOFs were an obvious starting point. NiDOBDC, MOF-5, and other members of this new class of framework-based sorbents are extremely porous. The frameworks can achieve surface areas ~10 times greater than activated charcoal. Further, the modular nature of the MOF synthesis method and their internal chemistry lets scientists build materials with a range of structures and properties.

"Interest in MOFs has exploded in the past decade," said Thallapally, who is also conducting research for DOE's Office of Nuclear Energy using these same materials.

At PNNL, Thallapally was working with MOFs for carbon sequestration, but saw more potential for the materials. He met with Grate to discuss MOF possibilities for security applications. They believed PNNL had the right capabilities to synthesize and characterize the necessary materials. They applied for and received PNNL funding to examine how well MOFs worked in xenon capture.

Previously, some MOFs proved disappointing. The materials worked well in the lab, but their framework structures were unstable under environmental conditions as vapors were sorbed and desorbed.

Working in PNNL's Sigma 5, the researchers tested three different xenon absorbers: NiDOBDC, activated charcoal, and a prototypical MOF, known as MOF-5. They found NiDOBDC takes up xenon about as well as activated charcoal and significantly better than MOF-5. NiDOBDC is superior at lower pressures. At 1 bar, the pressure at sea level, NiDOBDC was able to take up significant amounts of xenon and release all of it when the conditions were right. They also found that NiDOBDC was more selective than charcoal for xenon over krypton, which is fairly similar to xenon.

Thallapally and Grate are continuing to investigate MOFs, including NiDOBDC. Their work will include studies on scaling the materials to study their performance in real gas streams.

"While there isn't much you can do to improve charcoal, there is a world of potential in MOFs," said Thallapally. "The defined nanostructured framework gives you a lot of opportunities synthetically to add more functionality."

More information: PK Thallapally, et al. 2011. "Facile Xenon Capture and Release at Room Temperature using a Metal-Organic Framework: A Comparison with Activated Charcoal." Chemical Communications. DOI: 10.1039/C1CC14685H

Provided by Pacific Northwest National Laboratory (news : web)

Finding E. coli`s Achilles heel

E. coli are found naturally in human intestines and perform some important digestive duties. But a potentially deadly version is commonly found in spoiled or rotten food and attacks the human digestive system, causing food poisoning.

SFU and biochemistry (MBB) associate professor Mark Paetzel and his students Kelly Kim and Suraaj Aulakh have discovered how two proteins bind together in the outer membrane of E. coli.

The Journal of Biological Chemistry has just published their findings on-line in the paper Crystal structure of the ß-barrel assembly machinery BamCD complex.

Like many disease-causing forms of bacteria, E. coli bacteria are becoming increasingly resistant to conventional antibiotics. However, Paetzel, Kim (doctoral candidate) and Aulakh (master’s candidate) believe E. coli’s dependence on a factory-like machine in its outer membrane to keep it alive provides science with an untapped Achilles heel.

The trio has discovered how two proteins (BamC and BamD) in E. coli’s outer membrane bind together to help form what is known as the ß-barrel assembly machinery (BAM) complex.

Once up and running the complex ensures proper formation of proteins in the outer membrane, which serves as a protective barrier for E. coli. These proteins can function as foot soldiers that ensure E. coli’s survival by helping it to penetrate and attack its hosts, fight antibiotics and accomplish other tasks.

If Paetzel and his team isolate how the BAM complex’s other proteins bind together and collectively kick start the complex’s protein-assembly-mechanism they’ll have cornered E. coli’s Achilles heel.

“Being able to see and understand how this happens would enable us to design inhibitors to stymie the complex’s formation and startup,” adds Kim.

Says Aulakh: “It would be like watching a molecular movie in real time and designing a monkey wrench, effectively a new form of antibiotics, to shut down or cripple the complex before it starts functioning.”

The researchers hope the BAM complex could be a potential new drug target to help fight many diseases, such as meningitis and gonorrhea, which are also caused by BAM-containing bacteria.

Provided by Simon Fraser University