Combination of computational prescreening and experimental library construction can accelerate enzyme optimization by directed evolution

Chiral compounds can be produced efficiently by using biocatalysts. However, wild-type enzymes often do not meet the requirements of a production process, making optimization by rational design or directed evolution necessary. Here, we studied the lipase-catalyzed hydrolysis of the model substrate 1-(2-naphthyl)ethyl acetate both theoretically and experimentally. We found that a computational equivalent of alanine scanning mutagenesis based on QM/MM methodology can be applied to identify amino acid positions important for the activity of the enzyme. The theoretical results are consistent with concomitant experimental work using complete saturation mutagenesis and high-throughput screening of the target biocatalyst, a lipase from Bacillus subtilis. Both QM/MM-based calculations and molecular biology experiments identify histidine 76 as a residue that strongly affects the catalytic activity. The experiments demonstrate its important influence on enantioselectivity.     

Probing enzyme promiscuity of SGNH hydrolases

Several hydrolases of the SGNH superfamily, including the lipase SrLip from Streptomyces rimosus (Q93MW7), the acyl-CoA thioesterase I TesA from Pseudomonas aeruginosa (Q9HZY8) and the two lipolytic enzymes EstA (from P. aeruginosa, O33407) and EstP (from Pseudomonas putida, Q88QS0), were examined for promiscuity. These enzymes were tested against four chemically different classes of a total of 34 substrates known to be hydrolysed by esterases, thioesterases, lipases, phospholipases, Tweenases and proteases. Furthermore, they were also analysed with respect to their amino acid sequences and structural homology, and their phylogenetic relationship was determined. The Pseudomonas esterases EstA and EstP each have an N-terminal domain with catalytic activity together with a C-terminal autotransporter domain, and so the hybrid enzymes EstA(N)-EstP(C) and EstP(N)-EstA(C) were constructed by swapping the corresponding N- and C-terminal domains, and their hydrolytic activities were compared. Interestingly, substrate specificity and kinetic measurements indicated a significant influence of the autotransporter domains on the catalytic activities of these enzymes in solution. TesA, EstA and EstP were shown to function as esterases with different affinities and catalytic efficacies towards p-nitrophenyl butyrate. Of all the enzymes tested, only SrLip revealed lipase, phospholipase, esterase, thioesterase and Tweenase activities.     

Estimating Sediment Loadings in the South Saskatchewan River Catchment

Water Resources Management - In river catchments, sediment fluxes facilitate the transport of nutrients and pollutants and reduce water quality, potentially impacting water body health and altering...     

Functional cell-surface display of a lipase-specific chaperone

Lipases are important enzymes in biotechnology. Extracellular bacterial lipases from Pseudomonads and related species require the assistance of specific chaperones, designated "Lif" proteins (lipase specific foldases). Lifs, a unique family of steric chaperones, are anchored to the periplasmic side of the inner membrane where they convert lipases into their active conformation. We have previously shown that the autotransporter protein EstA from P. aeruginosa can be used to direct a variety of proteins to the cell surface of Escherichia coli. Here we demonstrate for the first time the functional cell-surface display of the Lif chaperone and FACS (fluorescence-activated cell sorting)-based analysis of bacterial cells that carried foldase-lipase complexes. The model Lif protein, LipH from P. aeruginosa, was displayed at the surface of E. coli cells. Surface exposed LipH was functional and efficiently refolded chemically denatured lipase. The foldase autodisplay system reported here can be used for a variety of applications including the ultrahigh-throughput screening of large libraries of foldase variants generated by directed evolution.     

Optochemical Control of Bacterial Gene Expression: Novel Photocaged Compounds for Different Promoter Systems

Photocaged compounds are applied for implementing precise, optochemical control of gene expression in bacteria. To broaden the scope of UV-light-responsive inducer molecules, six photocaged carbohydrates were synthesized and photochemically characterized, with the absorption exhibiting a red-shift. Their differing linkage through ether, carbonate, and carbamate bonds revealed that carbonate and carbamate bonds are convenient. Subsequently, those compounds were successfully applied in vivo for controlling gene expression in E. coli via blue light illumination. Furthermore, benzoate-based expression systems were subjected to light control by establishing a novel photocaged salicylic acid derivative. Besides its synthesis and in vitro characterization, we demonstrate the challenging choice of a suitable promoter system for light-controlled gene expression in E. coli. We illustrate various bottlenecks during both photocaged inducer synthesis and in vivo application and possibilities to overcome them. These findings pave the way towards novel caged inducer-dependent systems for wavelength-selective gene expression.     

Impact of morphological parameters on water quality variables of a regulated lowland river.

An efficient method to investigate which morphological changes have greatest impact on the water quality is to carry out a computer modelling exercise. In this study, three models, a hydrodynamic (DYNHYD), a eutrophication model (EUTRO), and a sediment and micro-pollutant transport model (TOXI), were coupled together in the High Level Architecture (HLA) platform. DYNHYD dynamically simulates the propagation of a wave through a river reach one-dimensionally by solving the full dynamic wave equation. Important parameters that describe the river morphology are the roughness coefficient and the weir discharge coefficient. EUTRO simulates the phytoplankton-nutrient dynamics in water bodies with variables and parameters that regulate the process in this cycle. Examples of some of the more important parameters are the oxygen reaeration, phytoplankton growth or nitrification rates. TOXI was implemented to simulate the transport of sediments and heavy metals in which the most important transformation process of the metals was sorption. It is the aim of this study to see the effect that hydrodynamic parameters such as weir discharge and roughness coefficients have on water quality constituents, such as chlorophyll-a, dissolved oxygen and levels of ammonium and dissolved zinc.     

Learning from directed evolution: theoretical investigations into cooperative mutations in lipase enantioselectivity

Molecular modeling with classical force-fields has been used to study the reactant complex and the tetrahedral intermediate in lipase-catalyzed ester hydrolysis in 20 enzyme/substrate combinations. The R and S enantiomers of alpha-methyldecanoic acid ester served as substrates for the wild-type lipase from Pseudomonas aeruginosa and nine selected mutants. After suitable preparation of initial structures from an available wild-type crystal structure, each system was subjected to 1 ns CHARMM force-field molecular dynamics simulations. The resulting geometric and energetic changes allow interpretation of some experimentally observed effects of mutations, particularly with regard to the "hot spots" at residues 155 and 162. The replacement S155F enhances S enantiopreference through a steric relay involving Leu162. The double mutation S53P + L162G improves S enantioselectivity by creating a new binding pocket for the S enantiomer with an additional stabilizing hydrogen bond to His83. The simulations provide insight into remote and cooperative effects of mutations.     

The Plant Sesquiterpene Nootkatone Efficiently Reduces Heterodera schachtii Parasitism by Activating Plant Defense

Plant parasitic nematodes, including the beet cyst nematode Heterodera schachtii, constitute a devastating problem for crops worldwide. The limited availability of sustainable management options illustrates the need for new eco-friendly control means. Plant metabolites represent an invaluable source of active compounds for the discovery of such novel antagonistic agents. Here, we evaluated the impact of eight plant terpenoids on the H. schachtii parasitism of Arabidopsis thaliana. None of the metabolites affected the plant development (5 or 10 ppm). Nootkatone decreased the number of adult nematodes on A. thaliana to 50%, with the female nematodes being smaller compared to the control. In contrast, three other terpenoids increased the parasitism and/or female size. We discovered that nootkatone considerably decreased the number of nematodes that penetrated A. thaliana roots, but neither affected the nematode viability or attraction to plant roots, nor triggered the production of plant reactive oxygen species or changed the plant’s sesquiterpene profile. However, we demonstrated that nootkatone led to a significant upregulation of defense-related genes involved in salicylic and jasmonic acid pathways. Our results indicate that nootkatone is a promising candidate to be developed into a novel plant protection agent acting as a stimulator of plant immunity against parasitic nematodes.