Zeroing in on more powerful enzymes for degrading persistent pollutants

Certain chemical components, like PCBs, PAHs, and CFCs, are toxic biosphere pollutants that are resistant to microbial degradation. Microbial catabolic enzymes are unable to effectively metabolize them. The results obtained by Professor Sylvestre and his colleagues open up new possibilities for boosting the effectiveness of these enzymes to oxidize such compounds.

Professor Sylvestre's research team has shown that it is possible to obtain more flexible mutant enzymes by replacing some of their amino acids. Moreover, they have updated a sophisticated mechanism that helps boost the enzyme's performance not only with regard to the natural substrate, but also any other substrates it can metabolize. As such, more effective new enzymes can be developed using genetic engineering.

"From a green chemistry perspective, the results of our research could allow us to apply these enzymes to biocatalysis processes to synthesize biologically active compounds (such as flavonoids) that have strong antioxidant properties," explained Professor Michel Sylvestre, also an enzyme engineering specialist.

More information: The results were published in the following works:

Mohammadi, M., Viger, J.F., Kumar, P., Barriault, D., Bolin, J. T., Sylvestre, M. 2011. "Retuning Rieske-type oxygenase to expand substrate range." J. Biol Chem. 286, 27612-27621. http://www.jbc.org … 932c85d04095

Kumar, P., Mohammadi, M., Viger, J.F., Barriault, D., Gomez-Gil, L., Eltis, L.D., Bolin, J. T., and Sylvestre, M. 2011. "Structural insight into the expanded PCB-degrading abilities of a biphenyl dioxygenase obtained by directed evolution." J. Mol. Biol. 405, 531-547. http://www.science … 28361001209X

Dhindwal, S., D. N. Patil, M. Mohammadi, M. Sylvestre, S. Tomar, and P. Kumar. 2011. "Biochemical studies and ligand bound structures of biphenyl dehydrogenase from Pandoraea pnomenusa strain B-356 reveal a basis for broad specificity of the enzyme." J. Biol. Chem. 286, 37011-37022. http://www.jbc.org … 932c85d04095

Provided by INRS

Carbon nanotube composites for enzymes and cosmetics

 Toyohashi Tech researchers develop a low cost and efficient method for producing electrically conducting composites based on electrostatic adsorption of CNTs onto resin and ceramic particles for applications including the production of enzymes and cosmetics.

Hiroyuki Muto and colleagues at Japan’s Toyohashi University of Technology (Toyohashi Tech) have developed an innovative method for producing CNT (carbon nano-tube) resin composite material   that only requires 1/100 of the conventional amount of CNT additive to produce electrical conductivity in the composite material.

In this method, CNTs were mixed in an electrolyte solution and added to the composite, where the CNTs were adsorbed onto the surfaces of the resin particles due to electrostatic adsorption.  This procedure enabled the production of electrical conducting composites by the addition of a small quantity CNTs.

Importantly, the electrical conductivity of the composite material was easily controlled by changing the amount of electrolyte added to the composite; namely, the concentration of CNTs adsorption onto the resin particles.

Notably, this approach enables significant reductions in both the production costs and the production time compared with conventional methods for manufacturing conductive resins.

The researchers are confident that adding particles with charged surfaces will enable the production of a wide range of composite materials such as metals, ceramics, and polymers.  This method is expected to find applications in the production of enzymes and cosmetics.

This work is supported by a Grant-in-Aid for Young Scientists at NEDO (New Energy and Industrial Technology Development Organization).

Salt-loving microbe provides new enzymes for the production of next-gen biofuels

In order to realize the full potential of advanced biofuels that are derived from non-food sources of lignocellulosic biomass—e.g., agricultural, forestry, and municipal waste, and crops such as poplar, switchgrass and miscanthus—new technologies that can efficiently and cost-effectively break down this biomass into simple sugars are required. Existing biomass pretreatment technologies are typically derived from the pulp and paper industry and rely on dilute acids and bases to break down the biomass. The treated biomass product is then exposed to biological catalysts, or enzymes, to liberate the sugars.

A new class of solvents, referred to as ionic liquids, have been reported to be much more efficient in treating the biomass and enhancing the yield of sugars liberated from it. While ionic liquids are useful for breaking down biomass, they can also hinder the ability of the cellulases (usually derived from fungi) used to produce sugars after pretreatment. Ionic liquids are a liquid form of salt that will inactivate enzymes by interfering with the folding of polypeptides—the building-blocks of proteins. To help identify new enzymes that are tolerant of ionic liquids, researchers from the U.S. Department of Energy (DOE) Joint Genome Institute (JGI) and the Joint BioEnergy Institute (JBEI) at DOE's Lawrence Berkeley National Laboratory are turning to those found in the complete genome sequences of halophilic (salt-tolerant) organisms.

As a test of this bioenergy-related application of DNA sequencing and enzyme discovery, researchers led by the Director of the DOE JGI, Eddy Rubin, and the Vice-President of the JBEI Deconstruction Division, Blake Simmons, employed a cellulose-degrading enzyme from a salt-tolerant microbe that was isolated from the Great Salt Lake. The microbe in question, Halorhabdus utahensis, is from the branch of the tree of life known as Archaea; H. utahensis was isolated from the natural environment at the Great Salt Lake and sequenced at the DOE JGI as part of the Genomic Encyclopedia of Bacteria and Archaea (GEBA) project.

"This is one of the only reports of salt-tolerant cellulases, and the only one that represents a true 'genome-to-function' relevant to ionic liquids from a halophilic environment," said Simmons of the study published June 30, 2011 in Green Chemistry. "This strategy enhances the possibility of identifying true obligatory halophilic enzymes." Such salt-tolerant enzymes, particularly cellulases, offer significant advantages for industrial utility over conventional enzymes.

In collaboration with Jerry Eichler from Ben Gurion University of the Negev in Israel they cloned and expressed a gene from H. utahensis in another haloarchaeal microbe, and were able to identify a salt-dependent enzyme that can tolerate high temperatures and is resistant to ionic liquids. "This project has established a very important link between genomic science and the realization of enzymes that can handle very demanding chemical environments, such as those present in a biorefinery," said Simmons.

The group plans to expand this research to develop a full complement of enzymes that is tailored for the ionic liquid process technology with the goal of demonstrating a complete biomass-to-sugar process, one they hope can enable the commercial viability of advanced biofuels.

Provided by DOE/Joint Genome Institute (news : web)

Spotlight on dynamic operation of enzymes

 Our world is unique in that living organisms can undergo complex chemical reactions quickly and precisely, and sequence them together. But how can proteins integral to life effectively hasten these reactions? Researchers from France provide new insight into how enzymes actually work. The study is presented in the journal PLoS Biology.

Scientists from the Institut des Sciences du Végétal (IVS) at the Centre National de la Recherche Scientifique (CNRS) in France, in cooperation with colleagues from the Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques (LCBPT), the Institut de Biochimie et Biophysique Moléculaire et Cellulaire (IBBMC) and the Laboratoire de Cristallographie et RMN Biologique, investigated the binding of a compound with therapeutic properties to its biological target.

Experts say specific macromolecules catalyse biochemical reactions and can be reused many times. The question, however, is if these proteins can speed up the reactions. What researchers know is that the enzyme must first recognise the substrate, which then comes into contact with certain chemical groups specific to it and is later transformed. The substrate is then favoured by the chemical environment that is established, and is linked to the deformations of molecular groups physically close to each other in space, according to the researchers.

So the macromolecular assembly reaches an ephemeral state that is highly reactive. Experts define this as the 'transition state'. What results is that the biochemical reaction is accelerated by a factor of several hundred billion.

Research from the 1950s unveiled the 'induced adjustment' model that had the substrate involved in changing the enzyme's form. Here, the small compound initially interacts with the enzyme, and this interaction triggers the conformational change of the macromolecule, which in turn enables the substrate's transformation.

In this latest study, the researchers used a therapeutic target enzyme, investigating a small compound mimicking the substrate that could bind strongly to the enzyme, and blocking its activity and exhibiting antibiotic, antineoplastic and herbicidal properties.

The team says an 'induced adjustment' stage is required to ensure the efficient binding of the compound to the target enzyme. In a nutshell, it is the tiny compound that brings about the conformational modification once attached to the enzyme.

By deriving the resolution of the fine structure of this enzyme from the Arabidopsis thaliana plant, the researchers effectively illustrated the interactions and conformations of each of the enzyme and substrate, at each stage of the reaction.

A hydrogen bond is formed, stabilising the enzyme-substrate complex in the transition state. This enables the enzymatic hydrolysis reaction to be carried out efficiently.

Thanks to their results, the researchers say this model can be used on all forms of the enzyme, especially those found in bacteria, which are targeted by antibiotics. The data also show the mechanism of how a therapeutic molecule can bind to its target, making it 'unbind' from it, and thus extending the drug's effect beyond the actual treatment, they say.

The results of this study can contribute to researchers' efforts to develop or improve the pharmacological properties of drug candidates.

More information: Fieulaine, S., et al. (2011) Trapping conformational states along ligand-binding dynamics of peptide deformylase: the impact of induced fit on enzyme catalysis. PLoS Biology. DOI:10.1371/journal.pbio.1001066

Provided by CORDIS

Enzymes turn vegetable oils into fuel through a flexible two-step process

Biodiesel is a promising future fuel, particularly because it can be made from a wide variety of renewable sources such as crude vegetable oils and waste fats produced by commercial kitchens. Conventional chemical processes for producing biodiesel, however, require pure and refined feedstock oils, thus negating any potential advantages. To get around this problem, Md. Mahabubur Rahman Talukder and co-workers at the A*STAR Institute of Chemical and Engineering Sciences have developed a two-step biocatalytic process that works well on all sorts of oils -- whether they are refined or not.

Making biodiesel involves breaking down vegetable oils through a reaction with methanol. Although some researchers have tried to use enzymes to catalyze this reaction, their efforts have seen little success because enzymes are deactivated when exposed to droplets of methanol. Various strategies have been developed to overcome this problem -- such as the gradual addition of methanol over time -- but none have proven suitable for industrial production.

Talukder’s approach involves splitting the process of biodiesel production into two separate steps. The first step involves hydrolyzing the oil, for which the researchers use a lipase enzyme called Candida rugosa. Vegetable oils consist of branched molecules known as triglycerides, which have three separate arms. Hydrolysis involves splitting off the arms so that each triglyceride is converted into three molecules of fatty acid.

The second step involves reacting the fatty acids with methanol to produce biodiesel. This step requires Novozym 435, an enzyme that is normally deactivated by methanol droplets. However, because methanol is much more soluble in fatty acids than in the triglyceride, all methanol added at this stage is dissolved in the fatty acids. Thus, because no methanol droplets form, the enzyme remains active.

“Avoiding enzyme deactivation is not the only advantage of our technique. The reaction between methanol and fatty acid progresses faster than the methanolysis of triglycerides,” says Talukder. “However, the key advantage is the feedstock flexibility.” In conventional chemical biodiesel production, impurities reduce the yield. This new approach, however, can accept feedstocks with any percentage of free fatty acids and water.

Talukder aims to make the process cheaper so that it can compete with chemical biodiesel production. “The lipase cost is one of the biggest challenges for the commercialization of the two-step process,” he says. “A low-cost lipase preparation technique is under consideration to improve the economic value of the process.” If successful, the technology could greatly help the environment and reduce the cost of fuel.

More information: Talukder, Md. M. R., et al. Two-step lipase catalysis for production of biodiesel. Biochemical Engineering Journal 49, 207–212 (2010) http://dx.doi.org/ … .2009.12.015

Provided by Agency for Science, Technology and Research (A*STAR)

Diamond X-rays used to discover tooth decay enzymes

Scientists using Diamond Light Source have made a breakthrough in the battle against tooth decay, with research published in the leading Journal of Molecular Biology (JMB) on 29 April 2011.

Researchers from the UK and Japan used the Diamond synchrotron in Oxfordshire and the Photon Factory in Tsukuba city, Japan, to solve the 3D structure of an enzyme that plays a key role in tooth decay caused by sugar.
Tooth decay can occur when a biofilm, or dental plaque as it is more commonly known, is formed by a large and sticky glucose polymer called glucan. The glucan biofilm contains bacteria and food debris and forms on the surface of the tooth. As they grow, the bacteria secrete acids which break down the tooth’s hard enamel on the surface. The structural information published in JMB provides a critical insight into how the enzyme ‘GTF-SI’, a glucansucrase, forms glucan, the sticky biofilm substance.

“With the use of the Diamond synchrotron and the Photon Factory we have been able to solve not only the structure of the enzyme alone but also its structure when bound to an existing inhibitor. Several inhibitors that prevent this type of enzyme forming glucan have been identified but to date there has been little structural information available. With the data we collected at Diamond and the Photon Factory, we now have a better understanding of how the enzyme functions and how it can be stopped. This structural information should be useful in the design of novel inhibitors that will prevent the biofilm formation by glucansucrases and reduce the risk of possible side effects such as hypoglycaemia. These novel inhibitors could be incorporated into toothpaste and mouthwash, making them more effective at preventing tooth decay," said Sohei Ito, Laboratory of Food Protein Engineering, University of Shizuoka in Japan, and lead researcher on the project.

The structural data collection at the Diamond synchrotron was carried out on the I02 Macromolecular Crystallography (MX) experimental station. Principal Beamline Scientist, Professor Thomas Sorensen, says, “Knowing the 3D structure of the enzyme is like knowing the shape of a lock you need to find a key for – it makes it much easier to find the right key that will fit. In this case, the inhibitor acts like the key, fitting into the lock in just the right way so that it can do its job.”

Sweet is an important favourable taste quality linked to food intake in humans and sucrose, the most common form of sugar, is the most highly consumed sweetener. But sucrose causes tooth decay, or dental caries as it is known. According to the World Oral Health Report 2003, dental caries is a major health problem in most industrialized countries, affecting 60-90% of school children and the vast majority of adults. If left untreated for a long period of time it can result in pain and tooth loss, and can lead to additional infections, periodontitis (gum disease), halitosis (bad breath) and in some cases even death by sepsis. Novel inhibitors open up the potential of reducing the risk of tooth decay by preventing the formation of dental plaque.
Diamond Light Source produces the extremely intense X-ray beams required for looking at the molecular interactions involved in a variety of biological processes. Advances in structural biology have accelerated greatly as a result of access to the synchrotron facilities that have been developed around the world in the past 25 years. Biologists have been swift to recognise the huge potential that lies behind understanding the multitude of processes that take place within living organisms at a molecular level. Researchers in the UK are at the forefront of this work and Diamond Light Source plays its part in providing cutting edge facilities for protein structure determination.
Diamond currently has five experimental stations dedicated to structural biology as well as an on-site Membrane Protein Laboratory. The work carried out at the synchrotron has the potential to affect our everyday lives. Previous breakthroughs using structural data from Diamond include gaining a better understanding of hypertension in the pre-natal condition pre-eclampsia, learning how a key tuberculosis drug is activated, understanding how bird flu can affect humans, and revealing the mechanism used by HIV to attack the body.

More information: ‘Crystal Structure of Glucansucrase from the Dental Caries Pathogen Streptococcus mutans’ Keisuke Ito, et al. Journal of Molecular Biology, Volume 408, Issue 2, Pages 177-378 (29 April 2011) http://dx.doi.org/ … 02.028 

Provided by Diamond Light Source

Positioning enzymes with ease

Virtually all processes in the human body rely on a unique class of proteins known as enzymes. To study them, scientists want to attach these molecules to surfaces and hold them fast, but this can often be a tricky undertaking.

Now Jinglin Fu and his colleagues at the Biodesign Institute at Arizona State University have developed a superior method for immobilizing enzymes on surfaces, deftly controlling their orientation, improving their efficiency and rendering them more stable. The group's results appear in today's advanced online issue of PLoS ONE.

Enzymes are essential for the normal functioning of cells, and are involved in tasks including cell regulation, metabolism and signal transduction. They are also necessary for muscle contraction and the transport of ions and other materials throughout the cytoskeleton.

Enzymes like amylases and proteases are central players in the digestive systems of many animals, breaking down starches and other large molecules into smaller parts that can be absorbed by the intestines. Herbivorous animals make use of the enzyme cellulose, to break down plant fiber. "No wonder enzyme function has been a topic of longstanding concern for biochemistry and medicine," says Fu.

Like other proteins, enzymes are composed of linear chains of amino acids. They can range from tens to thousands of amino acids in length. The job of the enzyme is to increase the rate of the desired reaction, without increasing the rate of undesired reactions. Here, a molecule known as the substrate interacts with a given enzyme to produce a product. Without enzymes, many reactions essential to living things could not proceed.

Such catalytic activity has also been adapted and broadly applied in the biomedical arena (especially for various diagnostic testing), as well for industrial applications ranging from photography to the brewing of beer.

Enzymes are also critical for the study of disease. Given their central role in maintaining homeostasis, any single enzyme aberration, including mutation, overproduction, underproduction or deletion can have dire consequences for health. Phenylketonuria, for example, is a disease linked with a single amino acid mutation in the enzyme phenylalanine hydroxylase. If untreated, the condition can lead to mental retardation. Malfunctioning of DNA repair enzymes is associated with a number of forms of cancer.

To properly study enzymes, particularly their catalytic activity, it is necessary to fix them in place on a surface. While researchers have used several techniques for enzyme immobilization, existing methods suffer from several shortcomings. Enzymes need to be properly oriented on the surface with respect to the molecule they are catalyzing in order to work properly. The non-specific binding of proteins can contaminate the reaction and lower or block its efficient progress. Finally, proteins are prone to becoming unfolded and deactivated over time—a process known as denaturation.

In the current study, Fu first generated a high-density array of peptides on a glass slide, each peptide composed of 20 randomly assembled amino acids. A specific enzyme, ß galactosidase, was then screened against this array. This method identified two peptides that covalently bound to the enzyme with high affinity, and these were used for the subsequent experiments.

When compared with low-affinity binding peptides and with preexisting surface immobilization techniques, the group found that the high affinity peptides not only were more effective at holding the enzyme in its proper orientation on the slide, they also produced higher specific activity in the enzyme. The enzyme was also less subject to denaturation, compared with controls.

In a further refinement of the technique, the group created mutations of the high affinity peptides, by deleting a single amino acid along the peptide's length and replacing it with a different amino acid. This procedure was repeated with all 20 amino acids in the peptide chain, with the resulting mutations once more screened against the ß galactosidase enzyme. The technique, known as single-point variant screening, improved both the binding affinity and specific activity of the bound enzyme.

"This development gives us a new tool, both for enhancing the function of surface bound enzymes, which are of ever-increasing importance to industry, and also for studying the interactions between multiple enzymes in a metabolic pathway," said Neal Woodburry, a co-author of the PLoS ONE study.

Provided by Arizona State University (news : web)

Nano fit-ness: Helping enzymes stay active and keep in shape

Proteins are critically important to life and the human body. They are also among the most complex molecules in nature, and there is much we still don't know or understand about them.

One key challenge is the stability of enzymes, a particular type of protein that speeds up, or catalyzes, chemical reactions. Taken out of their natural environment in the cell or body, enzymes can quickly lose their shape and denature. Everyday examples of enzymes denaturing include milk going sour, or eggs turning solid when boiled.

Rensselaer Polytechnic Institute Professor Marc-Olivier Coppens has developed a new technique for boosting the stability of enzymes, making them useful under a much broader range of conditions. Coppens confined lysozyme and other enzymes inside carefully engineered nanoscale holes, or nanopores. Instead of denaturing, these embedded enzymes mostly retained their 3-D structure and exhibited a significant increase in activity.

"Normally, when you put an enzyme on a surface, its activity goes down. But in this study, we discovered that when we put enzymes in nanopores – a highly controlled environment – the enzymatic activity goes up dramatically," said Coppens, a professor in the Department of Chemical and Biological Engineering at Rensselaer. "The enzymatic activity turns out to be very dependent on the local environment. This is very exciting."

Results of the study are detailed in the paper, "Effects of surface curvature and surface chemistry on the structure and activity of proteins adsorbed in nanopores," published last month by the journal Physical Chemistry Chemical Physics.

Researchers at Rensselaer and elsewhere have made important discoveries by wrapping enzymes and other proteins around nanomaterials. While this immobilizes the enzyme and often results in high stability and novel properties, the enzyme's activity decreases as it loses its natural 3-D structure.

Coppens took a different approach, and inserted enzymes inside nanopores. Measuring only 3-4 nanometers (nm) in size, the enzyme lysozyme fits snugly into a nanoporous material with well-controlled pore size between 5 nm and 12 nm. Confined to this compact space, the enzymes have a much harder time unfolding or wiggling around, Coppens said.

The discovery raises many questions and opens up entirely new possibilities related to biology, chemistry, medicine, and nanoengineering, Coppens said. He envisions this technology could be adapted to better control nanoscale environments, as well as increase the activity and selectivity of different enzymes. Looking forward, Coppens and colleagues will employ molecular simulations, multiscale modeling methods, and physical experiments to better understand the fundamental mechanics of confining enzymes inside nanopores.

More information: The paper may be viewed online at: http://dx.doi.org/10.1039/C0CP02273J

Provided by Rensselaer Polytechnic Institute (news : web)

Nano Fitness: Helping enzymes stay active and keep in shape

Proteins are critically important to life and the human body. They are also among the most complex molecules in nature, and there is much we still don't know or understand about them.

One key challenge is the stability of enzymes, a particular type of protein that speeds up, or catalyzes, chemical reactions. Taken out of their natural environment in the cell or body, enzymes can quickly lose their shape and denature. Everyday examples of enzymes denaturing include milk going sour, or eggs turning solid when boiled.

Rensselaer Polytechnic Institute Professor Marc-Olivier Coppens has developed a new technique for boosting the stability of enzymes, making them useful under a much broader range of conditions. Coppens confined lysozyme and other enzymes inside carefully engineered nanoscale holes, or nanopores. Instead of denaturing, these embedded enzymes mostly retained their 3-D structure and exhibited a significant increase in activity.

"Normally, when you put an enzyme on a surface, its activity goes down. But in this study, we discovered that when we put enzymes in nanopores -- a highly controlled environment -- the enzymatic activity goes up dramatically," said Coppens, a professor in the Department of Chemical and Biological Engineering at Rensselaer. "The enzymatic activity turns out to be very dependent on the local environment. This is very exciting."

Results of the study were published last month by the journal Physical Chemistry Chemical Physics.

Researchers at Rensselaer and elsewhere have made important discoveries by wrapping enzymes and other proteins around nanomaterials. While this immobilizes the enzyme and often results in high stability and novel properties, the enzyme's activity decreases as it loses its natural 3-D structure.

Coppens took a different approach, and inserted enzymes inside nanopores. Measuring only 3-4 nanometers (nm) in size, the enzyme lysozyme fits snugly into a nanoporous material with well-controlled pore size between 5 nm and 12 nm. Confined to this compact space, the enzymes have a much harder time unfolding or wiggling around, Coppens said.

The discovery raises many questions and opens up entirely new possibilities related to biology, chemistry, medicine, and nanoengineering, Coppens said. He envisions this technology could be adapted to better control nanoscale environments, as well as increase the activity and selectivity of different enzymes. Looking forward, Coppens and colleagues will employ molecular simulations, multiscale modeling methods, and physical experiments to better understand the fundamental mechanics of confining enzymes inside nanopores.

The study was co-authored by Lung-Ching Sang, a former Rensselaer graduate student in the Department of Chemical and Biological Engineering.

This research was supported by the National Science Foundation, via the Nanoscale Science and Engineering Center for Directed Assembly of Nanostructures at Rensselaer. The project was also supported by the International Center for Materials Nanoarchitectonics of the National Institute for Materials Science, Japan.

Story Source:

The above story is reprinted  from materials provided by Rensselaer Polytechnic Institute.

Journal Reference:

Lung-Ching Sang, Marc-Olivier Coppens. Effects of surface curvature and surface chemistry on the structure and activity of proteins adsorbed in nanopores. Physical Chemistry Chemical Physics, 2011; 13 (14): 6689 DOI: 10.1039/C0CP02273J

New insight into how 'tidying up' enzymes work

A new discovery about how molecules are broken down by the body, which will help pharmaceutical chemists design better drugs, has been made by researchers at the University of Bristol.

Working with Professor Jeremy Harvey and Professor Adrian Mulholland of Bristol's School of Chemistry, Dr Julianna Olah, an EU Marie Curie Fellow in Bristol at the time, studied a class of enzymes – cytochromes P450 – which play an important role in removing drug molecules from the body.

When a tablet of medicine is taken, the active molecules get absorbed into the bloodstream through the gut and make their way around the body, including to the cells in which they are intended to act; however, it's important they don't stay in the body forever. Enzymes (biological catalysts) help break them down to facilitate excretion.

The cytochromes P450 are a very important class of these 'tidying up' enzymes which have evolved to deal with all 'foreign' compounds that do not get broken down as part of normal metabolism (that is, any compounds which are not proteins, carbohydrates or lipids).

Mainly situated in the liver, the P450 enzymes help remove drug molecules by adding oxygen to them. This process usually works smoothly, but for some molecules, it can lead to oxygenated variants that are toxic. Other molecules are also able to interfere with the normal function of the P450 enzymes.

For these reasons, it is important to be able to understand how a given new molecule, considered for use as a medicine, will react with these enzymes. The Bristol researchers aimed to provide this understanding by modeling the reaction mechanism for interaction between one specific drug (dextromethorphan, a component of some cough syrups) and one P450 variant.

Professor Jeremy Harvey said: "Our calculations showed that the outcome of the oxygen transfer process (that is, which part of dextromethorphan oxygen gets added to) is affected by three factors.

"The first is the way in which the molecule fits into the enzyme ('docking'). The second is the intrinsic ability of each part of the molecule to accept oxygen. The third is how much each competing oxygen-delivery process is compatible with the shape of the enzyme pocket where the reaction occurs.

"While these first two factors were already known, the third was not. This discovery can help pharmaceutical chemists design new drug molecules with a better understanding of how they will be broken down in the body."

Provided by University of Bristol (news : web)

Tuned enzymes: Extra guest molecule in an enzyme's binding pocket enables methane oxidation

 Our fossil fuel reserves are limited. When they run out, we will not only be lacking in fuel, but chemical industry will lose its most important feedstock. In contrast, natural gas has barely been used as a raw material. If it were possible to efficiently convert methane, the main component of natural gas, into chemically useful materials like methanol, we would gain some time to make the transition to alternative sources of raw materials.

In the journal Angewandte Chemie, Manfred T. Reetz and a team at the Max Planck Institute for Carbon Research in Mülheim (Germany) have now introduced a new approach for the enzymatic production of methanol from methane. Their secret is the inclusion of an inert guest in the enzyme’s binding pocket in order to make it smaller so that it can effectively bind methane.

Methanol is a useful starting material for many chemical syntheses, and it can also be added to conventional fuels to drive fuel cells. Conventional processes for producing methanol from methane involve detours (synthesis gas), are markedly complex and energy intensive, and require high temperatures and pressures. Nature, on the other hand, has a much more elegant route: the enzyme methane monooxygenase does the job gently and efficiently. Unfortunately this is a very complex enzyme that cannot easily be produced and used in an artificial environment. The cytochrome P450 (CYP) family of enzymes could represent an alternative starting point. The main job of these enzymes is the oxidation of various substances produced by the body or introduced to it. In the reaction, carbon–hydrogen bonds are oxidized to make alcohol groups (–OH). The active component of these enzymes is a heme, an iron–porphyrin complex similar to that in our hemoglobin.

The problem is that the binding pocket of this enzyme is just too big to snugly bind and oxidize small molecules such as methane. Instead of trying to devise complex methods to create a suitable enzyme, Reetz and his co-workers came up with a clever trick: chemically “tuning” a CYP enzyme. The scientists added an additional guest into the binding pocket in order to make it smaller.

The natural substrates for CYP enzymes are fatty acids. As a guest molecule, the researchers chose a compound that resembles a fatty acid, a carbonic acid in which all of the hydrogen atoms in the hydrocarbon chain have been replaced with fluorine atoms. This type of molecule is as water-repellent as the original, but takes up more room. The fluorine atoms make it chemically inert so that it does not participate in any reactions. Like the molecule it is modeled on, this guest is able to bring the iron–heme complex of the enzyme into its catalytically active state (high-spin state). The significantly smaller binding pocket now allows methane to bind effectively so that it can be oxidized to methanol.

Says Reetz: “The road to success is still far for a technical implementation, yet, the concept opens up new perspectives for the development of further reactions, such as the oxidation of other chemical compounds.”

More information: Manfred T. Reetz, Tuning a P450 Enzyme for Methane Oxidation, Angewandte Chemie International Edition, http://dx.doi.org/ … ie.201006587