Search

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Multidimensional fitness landscapes provide insights into the molecular basis of laboratory and natural evolution. To date, such efforts usually focus on limited protein families and a single enzyme trait, with little concern about the relationship between protein epistasis and conformational dynamics. Here, we report a multiparametric fitness landscape for a cytochrome P450 monooxygenase that was engineered for the regio- and stereoselective hydroxylation of a steroid. We develop a computational program to automatically quantify non-additive effects among all possible mutational pathways, finding pervasive cooperative signs and magnitude epistasis on multiple catalytic traits. By using quantum mechanics and molecular dynamics simulations, we show that these effects are modulated by long-range interactions in loops, helices and β-strands that gate the substrate access channel allowing for optimal catalysis. Our work highlights the importance of conformational dynamics on epistasis in an enzyme involved in secondary metabolism and offers insights for engineering P450s., Connecting conformational dynamics and epistasis has so far been limited to a few proteins and a single fitness trait. Here, the authors provide evidence of positive epistasis on multiple catalytic traits in the evolution and dynamics of engineered cytochrome P450 monooxygenase, offering insights for in silico protein design.

P*-Mono- and P*,P*-bidentate phosphorodiamidites containing the (S a )-1,1′-binaphthyl core and 1,3,2-diazaphospholidine rings were synthesized. The use of these compounds in the rhodium-catalyzed asymmetric hydrogenation, as well as in the palladium- and platinum-catalyzed asymmetric alkylation and amination, resulted in the formation of the products with ee of up to 99%.

International audience; 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.

Iterative saturation mutagenesis (ISM) is a new and efficient method for the directed evolution of functional enzymes. It reduces the necessary molecular biological work and the screening effort drastically. It is based on a Cartesian view of the protein structure, performing iterative cycles of saturation mutagenesis at rationally chosen sites in an enzyme, a given site being composed of one, two or three amino acid positions. The basis for choosing these sites depends on the nature of the catalytic property to be improved, e.g., enantioselectivity, substrate acceptance or thermostability. In the case of thermostability, sites showing highest B-factors (available from X-ray data) are chosen. The pronounced increase in thermostability of the lipase from Bacillus subtilis (Lip A) as a result of applying ISM is illustrated here.

Saturation mutagenesis is a powerful technique for engineering proteins, metabolic pathways and genomes. In spite of its numerous applications, creating high-quality saturation mutagenesis libraries remains a challenge, as various experimental parameters influence in a complex manner the resulting diversity. We explore from the economical perspective various aspects of saturation mutagenesis library preparation: We introduce a cheaper and faster control for assessing library quality based on liquid media; analyze the role of primer purity and supplier in libraries with and without redundancy; compare library quality, yield, randomization efficiency and annealing bias using traditional and emergent randomization schemes based on mixtures of mutagenic primers; and establish a methodology for choosing the most cost-effective randomization scheme given the screening costs and other experimental parameters. We show that by carefully considering these parameters, laboratory expenses can be significantly reduced.

Chiral monophosphites, monophosphonites derived from BINOL are more effective ligandsin enantioselective rhodium-catalyzed hydrogenation of olefins, as compared to bidentate bis-phosphites.An appreciable increase in enantioselectivity is attained with the use of mixtures of two different monodentate phosphites and phosphonites in rhodium-catalyzed hydrogentation.

Racemic or enantiomerically pure alcohols can be converted with high yield into enantiopure chiral amines in a one-pot redox-neutral cascade process by the clever combination of an alcohol dehydrogenase and an appropriate amine dehydrogenase.

An in situ synchrotron radiation study of quasi-free five nanometer-sized palladium clusters during hydrogen absorption is combined with molecular dynamics simulations to investigate the structural development. In the diffraction patterns, strong intensity changes are found that provide evidence for a structural phase transformation that is significantly different from the α-\(\)-Pd-H bulk phase transition. The structural transition is reversible and driven by the hydrogen concentration. The intensity changes are consistent with a cubic-to-icosahedral structural phase transition obtained in molecular dynamical simulations using embedded-atom-method potentials.

Noble metal nanoparticle containing [Me]x-MCM-41 were synthesized using surfactant stabilised palladium, iridium and rhodium nanoparticles in the synthesis gel. The materials were characterised with XRD, ICP AES, TA, TEM and nitrogen sorption, which showed that the nanoparticles were present inside the pores of MCM-41. The [Me]x-MCM-41 were found to be active and selective catalysts in the hydrogenation of cyclic olefins such as cyclohexene, cyclooctene, cyclododecene and norbornene.

Directed evolution has emerged as a key technology to generate enzymes with new or improved properties that are of major importance to the biotechnology industry. A directed evolution approach starts with the identification of a target enzyme to be optimized and the cloning of the corresponding gene. An efficient expression system is needed before the target gene is subjected to random mutagenesis and/or in vitro recombination, thereby creating molecular diversity. Subsequently, improved enzyme variants are identified, preferably after being secreted into culture medium, by screening or selection for the desired property. The genes encoding the improved enzymes are then used to parent the next round of directed evolution. Enantioselectivity is a biocatalyst property of major biotechnological importance that is, however, difficult to deal with. We discuss recent examples of creating enantioselective biocatalysts by directed evolution.

The fluoride-catalysed hydrolysis of mixtures of CH3Si(OCH3)3 and Mg(OC2H5)2 in the presence of preformed nanosized R4N+Br−-stabilised Pd colloids results in the formation of micro/mesoporous hydrophobic sol–gel materials in which the Pd clusters are entrapped individually in the solid matrix. The materials are active catalysts in the hydrogenation of 1,5-cyclooctadiene.

The combination of molecular recognition, phase transfer catalysis and transition metal catalysis in one and the same water–soluble catalyst leads to new perspectives, including increased activity, novel substrate selectivity and the possibility of simple catalyst recovery. For example, Rh–complexes of β–cyclodextrin modified diphosphanes function as supramolecular catalysts in aqueous two–phase hydrogenation and hydroformylation. Metal–containing dendritic compounds constitute another new class of supramolecular catalysts which are easily recycled.

Pseudomonas aeruginosa secretes an extracellular lipase (EC 3.1.1.3), which has been isolated from culture media of either industrial fermentation of wild-type P. aeruginosa PAC1R or an overexpressing P. aeruginosa strain carrying a plasmid with the cloned lipase gene. Both culture supernatants contained enzymatically active lipase protein, as demonstrated by determination of hydrolytic activity using p-nitrophenylpalmitate and 1,2-O-dilauryl-rac-glycero-3-glutaric acid resorufin ester as substrates and analysis by sodium dodacyl sulphate/polyacrylamide electrophoresis and Western blotting. Immobilization by entrapment into chemically inert hydrophobic silica gels was tested with crude enzyme preparations. A matrix consisting of tetramethoxysilane and propyltrimethoxysilane at a molar ratio of 1 : 5 yielded the highest enzyme activity as determined by esterification of lauric acid with 1-octanol in isooctane. The biotechnological potential of P. aeruginosa lipase to catalyse the kinetic resolution of chiral compounds was tested by enantioselective acylation of two different model compounds, racemic 1-phenylethanol and 2-pentylamine. Both compounds were acylated with high efficiency giving enantiomeric excess rates of more than 99% for the alcohol and 96% for the amine with an average conversion rate of 50%. These results demonstrated that P. aeruginosa lipase is an extremely useful enzyme for application in synthetic organic chemistry.

The entrapment of lipases in hydrophobic sol-gel materials of RSi(OCH3)3 or mixtures of RSi(OCH3)3 and Si(OCH3)4 results in heterogeneous biocatalysts having dramatically enhanced enzyme activities as measured by the esterification of lauric acid by n-octanol in isooctane. These materials have been characterized by solid state NMR studies, revealing the degree of cross-linking. It is shown that this parameter generally does not correlate with relative enzyme activity. Likewise, the specific surface area or the pore size does not seem to be the decisive factor in determining the relative enzyme activities of the lipase-containing hybrid gels and the corresponding SiO2-gels obtained from Si(OCH3)4. Scanning electron microscopic studies (SEM) show that the hybrid gels all have a similar morphology. On the basis of these studies a model is proposed according to which most of the lipase enzyme is entrapped near the surface of the gel particles, where it is readily accessible by substrate molecules.

The electronic structure and heats of formation of several acyloxy cations and the isomeric dioxacyclopropyl cations have been determined by MINDO/2 calculations. The potential energy surface shows the cyclic isomer to represent the least energy species. The stabilizing effect of alkyl substituents in the latter has been traced to strong hyperconjugative interaction.