Engineering terpene synthases and their substrates for the biocatalytic production of terpene natural products and analogues.

Terpene synthases produce a wide number of hydrocarbon skeletons by controlling intramolecular rearrangements of allylic pyrophosphate subtrates reactive carbocation intermediates. Here we review recent research focused on engineering terpene synthases and modifying their substrates to rationally manipulate terpene catalyisis. Molecular dynamic simulations and solid state X-ray crystallography are powerful techniques to identify substrate binding modes, key active site residues for substrate folding, and the location of active site water.

Simulation-Guided Engineering Enables a Functional Switch in Selinadiene Synthase toward Hydroxylation.

Engineering sesquiterpene synthases to form predefined alternative products is a major challenge due to their diversity in cyclization mechanisms and our limited understanding of how amino acid changes affect the steering of these mechanisms. Here, we use a combination of atomistic simulation and site-directed mutagenesis to engineer a selina-4(15),7(11)-diene synthase (SdS) such that its final reactive carbocation is quenched by trapped active site water, resulting in the formation of a complex hydroxylated sesquiterpene (selin-7(11)-en-4-ol). Initially, the SdS G305E variant produced 20% selin-7(11)-en-4-ol.

Single Point Mutation Abolishes Water Capture in Germacradien-4-ol Synthase.

The high-fidelity sesquiterpene cyclase (-)-germacradien-4-ol synthase (GdolS) converts farnesyl diphosphate into the macrocyclic alcohol (-)-germacradien-4-ol. Site-directed mutagenesis was used to decipher the role of key residues in the water control mechanism. Replacement of Ala176, located in the G1/2 helix, with non-polar aliphatic residues of increasing size (valine, leucine, isoleucine and methionine) resulted in the accumulation of the non-hydroxylated products germacrene A and germacrene D.

Production of non-natural terpenoids through chemoenzymatic synthesis using substrate analogs.

Chemoenzymatic synthesis of non-natural terpenes using the promiscuous activity of terpene synthases allows for the expansion of the chemical space of terpenoids with potentially new bioactivities. In this report, we describe protocols for the preparation of a novel aphid attractant, (S)-14,15-dimethylgermacrene D, by exploiting the promiscuity of (S)-germacrene D synthase from Solidago canadensis and using an engineered biocatalytic route to convert prenols to terpenoids. The method uses a combination of five enzymes to carry out the preparation of terpenoid semiochemicals in two steps: (1) diphosphorylation of five or six carbon precursors (prenol, isoprenol and methyl-isoprenol) catalyzed by Plasmodium falciparum choline kinase and Methanocaldococcus jannaschii isopentenyl phosphate kinase to form DMADP, IDP and methyl-IDP, and (2) chain elongation and cyclization catalyzed by Geobacillus stearothermophilus (2E,6E)-farnesyl diphosphate synthase and S.

A Photoresponsive Homing Endonuclease for Programmed DNA Cleavage.

Homing endonucleases are used in a wide range of biotechnological applications including gene editing, in gene drive systems, and for the modification of DNA structures, arrays, and prodrugs. However, controlling nuclease activity and sequence specificity remain key challenges when developing new tools. Here a photoresponsive homing endonuclease was engineered for optical control of DNA cleavage by partitioning DNA binding and nuclease domains of the monomeric homing endonuclease I-TevI into independent polypeptide chains.

Active Site Loop Engineering Abolishes Water Capture in Hydroxylating Sesquiterpene Synthases.

Terpene synthases (TS) catalyze complex reactions to produce a diverse array of terpene skeletons from linear isoprenyl diphosphates. Patchoulol synthase (PTS) from converts farnesyl diphosphate into patchoulol. Using simulation-guided engineering, we obtained PTS variants that eliminate water capture.

Cryptic recessive lethality of a supergene controlling social organization in ants.

Supergenes are clusters of linked loci that control complex phenotypes, such as alternative forms of social organization in ants. Explaining the long-term maintenance of supergenes is challenging, particularly when the derived haplotype lacks homozygous lethality and causes gene drive. In the Alpine silver ant, Formica selysi, a large and ancient social supergene with two haplotypes, M and P, controls colony social organization.

Spatio-Temporal Photoactivation of Cytotoxic Proteins.

Protein therapeutics offer exquisite selectivity in targeting cellular processes and behaviors, but are rarely used against non-cell surface targets due to their poor cellular uptake. While cell-penetrating peptides can be used to deliver recombinant proteins to the cytosol, it is generally difficult to selectively deliver active proteins to target cells. Here, we report a recombinantly produced, intracellular protein delivery and targeting platform that uses a photocaged intein to regulate the spatio-temporal activation of protein activity in selected cells upon irradiation with light.

Bisphosphonate inhibitors of squalene synthase protect cells against cholesterol-dependent cytolysins.

Certain species of pathogenic bacteria damage tissues by secreting cholesterol-dependent cytolysins, which form pores in the plasma membranes of animal cells. However, reducing cholesterol protects cells against these cytolysins. As the first committed step of cholesterol biosynthesis is catalyzed by squalene synthase, we explored whether inhibiting this enzyme protected cells against cholesterol-dependent cytolysins.

Cryo-kinetics Reveal Dynamic Effects on the Chemistry of Human Dihydrofolate Reductase.

Effects of isotopic substitution on the rate constants of human dihydrofolate reductase (HsDHFR), an important target for anti-cancer drugs, have not previously been characterized due to its complex fast kinetics. Here, we report the results of cryo-measurements of the kinetics of the HsDHFR catalyzed reaction and the effects of protein motion on catalysis. Isotopic enzyme labeling revealed an enzyme KIE (k /k ) close to unity above 0 °C; however, the enzyme KIE was increased to 1.

Redesigning the Molecular Choreography to Prevent Hydroxylation in Germacradien-11-ol Synthase Catalysis.

Natural sesquiterpene synthases have evolved to make complex terpenoids by quenching reactive carbocations either by proton transfer or by hydroxylation (water capture), depending on their active site. Germacradien-11-ol synthase (Gd11olS) from catalyzes the cyclization of farnesyl diphosphate (FDP) into the hydroxylated sesquiterpene germacradien-11-ol. Here, we combine experiment and simulation to guide the redesign of its active site pocket to avoid hydroxylation of the product.

Electric Field Measurements Reveal the Pivotal Role of Cofactor-Substrate Interaction in Dihydrofolate Reductase Catalysis.

The contribution of ligand-ligand electrostatic interaction to transition state formation during enzyme catalysis has remained unexplored, even though electrostatic forces are known to play a major role in protein functions and have been investigated by the vibrational Stark effect (VSE). To monitor electrostatic changes along important steps during catalysis, we used a nitrile probe (T46C-CN) inserted proximal to the reaction center of three dihydrofolate reductases (DHFRs) with different biophysical properties, DHFR (EcDHFR), its conformationally impaired variant (EcDHFR-S148P), and DHFR (BsDHFR). Our combined experimental and computational approach revealed that the electric field projected by the substrate toward the probe negates those exerted by the cofactor when both are bound within the enzymes.

Accelerating Biphasic Biocatalysis through New Process Windows.

Process intensification through continuous flow reactions has increased the production rates of fine chemicals and pharmaceuticals. Catalytic reactions are accelerated through an unconventional and unprecedented use of a high-performance liquid/liquid counter current chromatography system. Product generation is significantly faster than in traditional batch reactors or in segmented flow systems, which is exemplified through stereoselective phase-transfer catalyzed reactions.

Selective Hydroboration-Oxidation of Terminal Alkenes under Flow Conditions.

An efficient flow process for the selective hydroboration and oxidation of different alkenes using 9-borabicyclo(3.3.1)nonane (9-BBN) allows facile conversion in high productivity (1.

Modular Chemoenzymatic Synthesis of Terpenes and their Analogues.

Non-natural terpenoids offer potential as pharmaceuticals and agrochemicals. However, their chemical syntheses are often long, complex, and not easily amenable to large-scale production. Herein, we report a modular chemoenzymatic approach to synthesize terpene analogues from diphosphorylated precursors produced in quantitative yields.

An unexpected co-crystal structure of the calpain PEF(S) domain with Hfq reveals a potential chaperone function of Hfq.

Calpain is a Ca-activated, heterodimeric cysteine protease consisting of a large catalytic subunit and a small regulatory subunit. Dysregulation of this enzyme is involved in a range of pathological conditions such as cancer, Alzheimer's disease and rheumatoid arthritis, and thus calpain I is a drug target with potential therapeutic applications. Difficulty in the production of this enzyme has hindered structural and functional investigations in the past, although heterodimeric calpain I can be generated by Escherichia coli expression in low yield.

Harnessing enzyme plasticity for the synthesis of oxygenated sesquiterpenoids.

8-Methoxy-γ-humulene, ()-8-methoxy-β-farnesene, 12-methoxy-β-sesquiphellandrene and 12-methoxyzingiberene can be synthesised in amorphadiene synthase-catalysed reactions from 8- and 12-methoxyfarnesyl diphosphates due to the highly plastic yet tightly controlled carbocationic chemistry of this sesquiterpene cyclase.

"Dark" Singlet Oxygen Made Easy.

An operationally simple continuous flow generator of "dark" singlet oxygen has been developed. The singlet oxygen was efficiently reacted with several chemical traps to give the corresponding oxygenated products in high yields. The developed "dark" singlet oxygen generator has been successfully applied in the synthesis of the antimalarial drug artemisinin.

Why are some Enzymes Dimers? Flexibility and Catalysis in Dihydrofolate Reductase.

Dihydrofolate Reductase from (TmDFHFR) is a dimeric thermophilic enzyme that catalyzes the hydride transfer from the cofactor NADPH to dihydrofolate less efficiently than other DHFR enzymes, such as the mesophilic analogue DHFR (EcDHFR). Using QM/MM potentials we show that the reduced catalytic efficiency of TmDHFR is most likely due to differences in the amino acid sequence that stabilize the M20 loop in an open conformation, which prevents the formation of some interactions in the transition state and increases the number of water molecules in the active site. However, dimerization provides two advantages to the thermophilic enzyme; it protects its structure against denaturation by reducing thermal fluctuations and it provides a less negative activation entropy, toning down the increase of the activation free energy with temperature.

A Noncanonical Chromophore Reveals Structural Rearrangements of the Light-Oxygen-Voltage Domain upon Photoactivation.

Light-oxygen-voltage (LOV) domains are increasingly used to engineer photoresponsive biological systems. While the photochemical cycle is well documented, the allosteric mechanism by which formation of a cysteinyl-flavin adduct leads to activation is unclear. Via replacement of flavin mononucleotide (FMN) with 5-deazaflavin mononucleotide (5dFMN) in the Aureochrome1a (Au1a) transcription factor from Ochromonas danica, a thermally stable cysteinyl-5dFMN adduct was generated.

Cell-penetrating peptide sequence and modification dependent uptake and subcellular distribution of green florescent protein in different cell lines.

Protein therapy holds great promise for treating a variety of diseases. To act on intracellular targets, therapeutic proteins must cross the plasma membrane. This has previously been achieved by covalent attachment to a variety of cell-penetrating peptides (CPPs).