Enlightening the Path to Protein Engineering: Chemoselective Turn-On Probes for High-Throughput Screening of Enzymatic Activity.

Many successful stories in enzyme engineering are based on the creation of randomized diversity in large mutant libraries, containing millions to billions of enzyme variants. Methods that enabled their evaluation with high throughput are dominated by spectroscopic techniques due to their high speed and sensitivity. A large proportion of studies relies on fluorogenic substrates that mimic the chemical properties of the target or coupled enzymatic assays with an optical read-out that assesses the desired catalytic efficiency indirectly.

Mutations Increasing Cofactor Affinity, Improve Stability and Activity of a Baeyer-Villiger Monooxygenase.

The typically low thermodynamic and kinetic stability of enzymes is a bottleneck for their application in industrial synthesis. Baeyer-Villiger monooxygenases, which oxidize ketones to lactones using aerial oxygen, among other activities, suffer particularly from these instabilities. Previous efforts in protein engineering have increased thermodynamic stability but at the price of decreased activity.

Storing and Reading Information in Mixtures of Fluorescent Molecules.

The rapidly increasing use of digital technologies requires the rethinking of methods to store data. This work shows that digital data can be stored in mixtures of fluorescent dye molecules, which are deposited on a surface by inkjet printing, where an amide bond tethers the dye molecules to the surface. A microscope equipped with a multichannel fluorescence detector distinguishes individual dyes in the mixture.

A click-flipped enzyme substrate boosts the performance of the diagnostic screening for Hunter syndrome.

We report on the unexpected finding that click modification of iduronyl azides results in a conformational flip of the pyranose ring, which led to the development of a new strategy for the design of superior enzyme substrates for the diagnostic assaying of iduronate-2-sulfatase (I2S), a lysosomal enzyme related to Hunter syndrome. Synthetic substrates are essential in testing newborns for metabolic disorders to enable early initiation of therapy. Our click-flipped iduronyl triazole showed a remarkably better performance with I2S than commonly used -iduronates.

Analysis of Powders Containing Illicit Drugs Using Magnetic Levitation.

Magneto-Archimedes levitation (MagLev) enables the separation of powdered mixtures of illicit drugs (cocaine, methamphetamine, heroin, fentanyl, and its analogues), adulterants, and diluents based on density, and allows the presumptive identification of individual components. Small samples (mass <50 mg), with low concentrations of illicit drugs, present a particular challenge to analysis for forensic chemists. The MagLev device, a cuvette containing a solution of paramagnetic gadolinium(III) chelate in a non-polar solvent, placed between two like-poles-facing NdFeB magnets, allowed separation of seven relevant compounds simultaneously.

Storage of Information Using Small Organic Molecules.

Although information is ubiquitous, and its technology arguably among the highest that humankind has produced, its very ubiquity has posed new types of problems. Three that involve storage of information (rather than computation) include its usage of energy, the robustness of stored information over long times, and its ability to resist corruption through tampering. The difficulty in solving these problems using present methods has stimulated interest in the possibilities available through fundamentally different strategies, including storage of information in molecules.

A Common Mechanism Links Activities of Butyrate in the Colon.

Two biological activities of butyrate in the colon (suppression of proliferation of colonic epithelial stem cells and inflammation) correlate with inhibition of the activity of histone deacetylases. Cellular and biochemical studies of molecules similar in structure to butyrate, but different in molecular details (functional groups, chain-length, deuteration, oxidation level, fluorination, or degree of unsaturation), demonstrated that these activities were sensitive to molecular structure, and were compatible with the hypothesis that butyrate acts by binding to the Zn in the catalytic site of histone deacetylases. Structure-activity relationships drawn from a set of 36 compounds offer a starting point for the design of new compounds targeting the inhibition of histone deacetylases.

The Molecular Origin of Enthalpy/Entropy Compensation in Biomolecular Recognition.

Biomolecular recognition can be stubborn; changes in the structures of associating molecules, or the environments in which they associate, often yield compensating changes in enthalpies and entropies of binding and no net change in affinities. This phenomenon-termed enthalpy/entropy (H/S) compensation-hinders efforts in biomolecular design, and its incidence-often a surprise to experimentalists-makes interactions between biomolecules difficult to predict. Although characterizing H/S compensation requires experimental care, it is unquestionably a real phenomenon that has, from an engineering perspective, useful physical origins.

Fails to Efficiently Maintain the Activity of an Important Flavin Monooxygenase in Recombinant Overexpression.

This paper describes the measurement and analysis of activity and stability of cyclohexanone monooxygenase from sp. NCIMB 9871 (CHMO), a model Baeyer-Villiger monooxygenase, in the recombinant host . This enzyme was often described as poorly stable , and has recently been found to deactivate rapidly in the absence of its essential cofactors and antioxidants.

Cloning and characterization of the Type I Baeyer-Villiger monooxygenase from Leptospira biflexa.

Baeyer-Villiger monooxygenases are recognized by their ability and high selectivity as oxidative biocatalysts for the generation of esters or lactones using ketones as starting materials. These enzymes represent valuable tools for biooxidative syntheses since they can catalyze reactions that otherwise involve strong oxidative reagents. In this work, we present a novel enzyme, the Type I Baeyer-Villiger monooxygenase from Leptospira biflexa.

Non-hazardous biocatalytic oxidation in Nylon-9 monomer synthesis on a 40 g scale with efficient downstream processing.

This paper describes the development of a biocatalytic process on the multi-dozen gram scale for the synthesis of a precursor to Nylon-9, a specialty polyamide. Such materials are growing in demand, but their corresponding monomers are often difficult to synthesize, giving rise to biocatalytic approaches. Here, we implemented cyclopentadecanone monooxygenase as an Escherichia coli whole-cell biocatalyst in a defined medium, together with a substrate feeding-product removal concept, and an optimized downstream processing (DSP).

Redesign of water networks for efficient biocatalysis.

Herein we highlight recent findings on the importance of water networks in proteins, and their redesign and reconfiguration as a new engineering strategy to generate enzymes with modulated binding affinity and improved catalytic versatility. Traditionally, enzyme engineering and drug design have focused on tailoring direct and favorable interactions between protein surfaces and ligands/transition states to achieve stronger binding, or an accelerated manufacturing of medicines, biofuels, fine chemicals and materials. In contrast, the opportunity to relocate water molecules in solvated binding pockets by protein design to improve overall energetics remains essentially unexplored, and fundamental understanding of the elusive processes involved is poor.

First chemo-enzymatic synthesis of the ()-Taniguchi lactone and substrate profiles of CAMO and OTEMO, two new Baeyer-Villiger monooxygenases.

Abstract: This study investigates the substrate profile of cycloalkanone monooxygenase and 2-oxo-Δ-4,5,5-trimethylcyclopentenylacetyl-coenzyme A monooxygenase, two recently discovered enzymes of the Baeyer-Villiger monooxygenase family, used as whole-cell biocatalysts. Biooxidations of a diverse set of ketones were performed on analytical scale: desymmetrization of substituted prochiral cyclobutanones and cyclohexanones, regiodivergent oxidation of terpenones and bicyclic ketones, as well as kinetic resolution of racemic cycloketones. We demonstrated the applicability of the title enzymes in the enantioselective synthesis of ()-(-)-Taniguchi lactone, a building block for the preparation of various natural product analogs such as ent-quinine.

Non-hazardous Baeyer-Villiger oxidation of levulinic acid derivatives: alternative renewable access to 3-hydroxypropionates.

Baeyer-Villiger monooxygenases catalyze the energetically challenging oxidation of levulinates (4-oxopentanoates) to 3-hydroxypropionic acid (3-HPA) derivates under ambient conditions, replacing propellant-grade H2O2 with aerial oxygen as the oxidant. This reaction enables a new pathway to a platform for chemical 3-HPA, an important intermediate in the non-petrol based production of a variety of bulk chemicals (acrylates, malonates, 1,3-propanediol).

Extending the substrate scope of a Baeyer-Villiger monooxygenase by multiple-site mutagenesis.

Baeyer-Villiger monooxygenase-catalysed reactions are attractive for industrial processes. Here we report on expanding the substrate scope of phenylacetone monooxygenase (PAMO). In order to introduce activity on alicyclic ketones in PAMO, we generated and screened a library of 1,500 mutants.

Baeyer-Villiger monooxygenases in aroma compound synthesis.

Baeyer-Villiger monooxygenases (BVMOs) are presented as highly selective and efficient biocatalysts for the synthesis of aroma lactones via kinetic resolution of 2-substituted cycloketones, exemplified with two δ-valerolactones, the jasmine lactones and their ε-caprolactone homologs. Analytical scale screens of our BVMO library ensued by preparative whole-cell biotransformations led to the identification of two enzymes (cyclohexanone monooxygenase from Arthrobacter BP2 and cyclododecanone monooxygenase from Rhodococcus SC1) perfectly suited for the task at hand: easily accessible racemic starting materials were bio-oxidized to almost enantiopure ketones and lactones in good yields (48-74%) and optical purities (ee 93% to >99%, E>100).