Calcium-dependent exocytosis of synaptic vesicles mediates the release of neurotransmitters. Important proteins in this process have been identified such as the SNAREs, synaptotagmins, complexins, Munc18 and Munc13. Structural and functional studies have yielded a wealth of information about the physiological role of these proteins. However, it has been surprisingly difficult to arrive at a unified picture of the molecular sequence of events from vesicle docking to calcium-triggered membrane fusion. Using mainly a biochemical and biophysical perspective, we briefly survey the molecular mechanisms in an attempt to functionally integrate the key proteins into the emerging picture of the neuronal fusion machine.

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Molecular machines governing exocytosis of synaptic vesicles

Phthalocyanine Metal Complexes in Catalysis

Coordinatively unsaturated (CUS) iron sites are highly active in catalytic oxidation reactions; however, maintaining the CUS structure of iron during heterogeneous catalytic reactions is a great challenge. Here, we report a strategy to stabilize single-atom CUS iron sites by embedding highly dispersed FeN4 centers in the graphene matrix. The atomic structure of FeN4 centers in graphene was revealed for the first time by combining high-resolution transmission electron microscopy/high-angle annular dark-field scanning transmission electron microscopy with low-temperature scanning tunneling microscopy. These confined single-atom iron sites exhibit high performance in the direct catalytic oxidation of benzene to phenol at room temperature, with a conversion of 23.4% and a yield of 18.7%, and can even proceed efficiently at 0°C with a phenol yield of 8.3% after 24 hours. Both experimental measurements and density functional theory calculations indicate that the formation of the Fe═O intermediate structure is a key step to promoting the conversion of benzene to phenol. These findings could pave the way toward highly efficient nonprecious catalysts for low-temperature oxidation reactions in heterogeneous catalysis and electrocatalysis.

A single iron site confined in a graphene matrix for the catalytic oxidation of benzene at room temperature.

The general principles of green chemistry guide the design of environmentally benign products and processes. It is known that the efficient utilization of carbon resources and carbon recycling are of great importance for the sustainable development of our society, and there are many related challenging scientific issues to be solved. Herein we attempt a holistic view on carbon resource processing, utilization, and recycling, and we call this “green carbon science”. We will discuss the definition and the four key principles of green carbon science and highlight some related fields including petroleum refining, the production of liquid fuels and chemicals from coal and methane, and transformations of CO2 and biomass into fuels and chemicals.

Green Carbon Science: Scientific Basis for Integrating Carbon Resource Processing, Utilization, and Recycling

Enzymes as catalysts in synthetic organic chemistry gained importance in the latter half of the 20th century, but nevertheless suffered from two major limitations. First, many enzymes were not accessible in large enough quantities for practical applications. The advent of recombinant DNA technology changed this dramatically in the late 1970s. Second, many enzymes showed a narrow substrate scope, often poor stereo- and/or regioselectivity and/or insufficient stability under operating conditions. With the development of directed evolution beginning in the 1990s and continuing to the present day, all of these problems can be addressed and generally solved. The present Perspective focuses on these and other developments which have popularized enzymes as part of the toolkit of synthetic organic chemists and biotechnologists. Included is a discussion of the scope and limitation of cascade reactions using enzyme mixtures in vitro and of metabolic engineering of pathways in cells as factories for the production o...

Biocatalysis in Organic Chemistry and Biotechnology: Past, Present, and Future

A current challenge in synthetic organic chemistry is the development of methods that allow the regio- and stereoselective oxidative C-H activation of natural or synthetic compounds with formation of the corresponding alcohols. Cytochrome P450 enzymes enable C-H activation at non-activated positions, but the simultaneous control of both regio- and stereoselectivity is problematic. Here, we demonstrate that directed evolution using iterative saturation mutagenesis provides a means to solve synthetic problems of this kind. Using P450 BM3(F87A) as the starting enzyme and testosterone as the substrate, which results in a 1:1 mixture of the 2β- and 15β-alcohols, mutants were obtained that are 96-97% selective for either of the two regioisomers, each with complete diastereoselectivity. The mutants can be used for selective oxidative hydroxylation of other steroids without performing additional mutagenesis experiments. Molecular dynamics simulations and docking experiments shed light on the origin of regio- and stereoselectivity.

Regio- and stereoselectivity of P450-catalysed hydroxylation of steroids controlled by laboratory evolution

Munc18–1, a protein essential for regulated exocytosis in neurons and neuroendocrine cells, belongs to the family of Sec1/Munc18-like (SM) proteins. In vitro, Munc18–1 forms a tight complex with the SNARE syntaxin 1, in which syntaxin is stabilized in a closed conformation. Since closed syntaxin is unable to interact with its partner SNAREs SNAP-25 and synaptobrevin as required for membrane fusion, it has hitherto not been possible to reconcile binding of Munc18–1 to syntaxin 1 with its biological function. We now show that in intact and exocytosis-competent lawns of plasma membrane, Munc18–1 forms a complex with syntaxin that allows formation of SNARE complexes. Munc18–1 associated with membrane-bound syntaxin 1 can be effectively displaced by adding recombinant synaptobrevin but not syntaxin 1 or SNAP-25. Displacement requires the presence of endogenous SNAP-25 since no displacement is observed when chromaffin cell membranes from SNAP-25–deficient mice are used. We conclude that Munc18–1 allows for the formation of a complex between syntaxin and SNAP-25 that serves as an acceptor for vesicle-bound synaptobrevin and that thus represents an intermediate in the pathway towards exocytosis.

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Munc18-bound syntaxin readily forms SNARE complexes with synaptobrevin in native plasma membranes.

Cytochrome P450 (CYP) enzymes are heme-dependent monooxygenases that catalyze the oxidation of C H bonds of endogenous and exogenous organic compounds with formation of the respective alcohols. The mechanism involves the intermediacy of a high-spin oxyferryl porphyrin radical cation which abstracts a hydrogen atom from the substrate, and the short-lived alkyl radical then undergoes C Obond formation. The binding pockets of CYPs are relatively large, therefore small compounds do not have a statistically high enough probability of being properly oriented near the oxyferryl moiety for rapid oxidation to occur; additionally there are other effects that slow down or prevent catalysis. A notorious challenge is the oxidation of methane to methanol by chemical catalysis or using enzymes of the type methane monooxygenases (MMOs). It is not only the smallest alkane, but also has the strongest C H bond (104 kcalmol ). Although CYPs represent a superfamily of monooxygenases, none have been shown to accept methane, whereas MMOs are complex enzymes (many membrane bound) that have not been expressed in heterologous hosts in any significant quantities, among other problems. Herein we show that chemical tuning of a CYP, which is based on guest/host activation using perfluoro carboxylic acids as chemically inert guests, activates the enzyme for oxidation of not only medium-sized alkanes such as n-hexane, but also of small gaseous molecules such as propane and even methane as the ultimate challenge. In the present study we chose, for practical reasons, the enzyme P450 BM3 (CYP102A1) from Bacillus megaterium, which is a self-sufficient fusion protein composed of a P450 monooxygenase and an NADPH diflavin reductase. Several crystal structures of this CYP harboring a fatty acid or fatty acid derived inhibitors, as well in the absence of such compounds have been published. To engineer mutants of P450 BM3 and of other CYPs for enhanced activity and selectivity toward a variety of different compounds, including such difficult substrates as small alkanes, rational design as well as directed evolution have proven to be successful to some extent. For example, P450 BM3 variants characterized by numerous point mutations were obtained in extensive laboratory evolution, and showed for the first time notable activity toward propane by formation of the respective alcohols (2-propanol/1-propanol= 9:1); however, the ethane to ethanol conversion remains problematic and methane oxidation has not been achieved to date. Higher activity in ethane oxidation was accomplished using mutants of P450cam, but here again methane oxidation was not reported. Our chemical approach involves a chemically inert compound that serves as a guest in the binding pocket of P450 BM3, thereby filling the space and reducing the translational freedom of small alkanes or of any other substrate. On the basis of previous reports involving CYPs harboring various substrates, such guest/host interactions can be expected to induce other modes of activation effects as well, specifically water displacement at the Fe/heme site accompanied by a change in the electronic state from the inactive low-spin state to the catalytically active high-spin states. Moreover, many studies have shown that P450 enzymes and mutants thereof can harbor two different substrates simultaneously, thus leading to cooperative effects; one example is lauric acid and palmitic acid in which cooperativity has been demonstrated by isotope labeling experiments. In yet another study regarding the metabolism of bilirubin, the addition of lauric acid or the perfluorinated analogue was reported to facilitate NADPH oxidation and substrate degradation, a finding that has implications for the treatment of jaundice, uroporphyria, and possibly cancer. It has also been shown for the case of a distantly related H2O2dependent P450 enzyme that its peroxidase activity can be influenced by the addition of fatty acids, wherein increased or decreased activity is observed depending upon their chain length. In our endeavor we were guided by the binding mode of the natural substrates, fatty acids, of P450 BM3. The binding includes H-bonds originating from their carboxy function and residues Arg 47 and Tyr 51, as well as hydrophobic interactions. The use of perfluoro carboxylic acids such as 1a–h as chemically inert, yet activating guests was therefore envisioned, because perfluoro alkyl groups are known to be resistant to oxidation while having a hydrophobic character. Moreover, it is known that a CF3 residue is sterically comparable to a CH(CH3)2 group, [11a] which means that a perfluoro fatty acid fills much more space in a P450 binding pocket than a traditional fatty acid, and can additionally induce the crucial low-spin to high-spin conversion of Fe/heme. In exploratory studies, the oxidation of n-octane and n-hexane as well as isomers thereof was studied using P450 [*] Dr. F. E. Zilly, Dr. J. P. Acevedo, Dr. W. Augustyniak, A. Deege, U. W. H usig, Prof. M. T. Reetz Max-Planck-Institut f r Kohlenforschung Kaiser-Wilhelm-Platz 1, 45470 M lheim an der Ruhr (Germany) reetz@mpiso-muelheim.mpg.de

Tuning a p450 enzyme for methane oxidation

Saturation mutagenesis constitutes a powerful method in the directed evolution of enzymes. Traditional protocols of whole plasmid amplification such as Stratagene’s QuikChange™ sometimes fail when the templates are difficult to amplify. In order to overcome such restrictions, we have devised a simple two-primer, two-stage polymerase chain reaction (PCR) method which constitutes an improvement over existing protocols. In the first stage of the PCR, both the mutagenic primer and the antiprimer that are not complementary anneal to the template. In the second stage, the amplified sequence is used as a megaprimer. Sites composed of one or more residues can be randomized in a single PCR reaction, irrespective of their location in the gene sequence.The method has been applied to several enzymes successfully, including P450-BM3 from Bacillus megaterium, the lipases from Pseudomonas aeruginosa and Candida antarctica and the epoxide hydrolase from Aspergillus niger. Here, we show that megaprimer size as well as the direction and design of the antiprimer are determining factors in the amplification of the plasmid. Comparison of the results with the performances of previous protocols reveals the efficiency of the improved method.

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Improved PCR method for the creation of saturation mutagenesis libraries in directed evolution: application to difficult-to-amplify templates

https://www.rsc.org/suppdata/CC/b9/b913863c/b913863c.pdf

Felipe E. Zilly

Papers

Regio- and stereoselectivity of P450-catalysed hydroxylation of steroids controlled by laboratory evolution

Munc18-bound syntaxin readily forms SNARE complexes with synaptobrevin in native plasma membranes.

Tuning a p450 enzyme for methane oxidation

Improved PCR method for the creation of saturation mutagenesis libraries in directed evolution: application to difficult-to-amplify templates

Deazaflavins as mediators in light-driven cytochrome P450 catalyzed hydroxylations