36 research outputs found
How a 10-epi-cubebol Synthase Avoids Premature Reaction Quenching to Form a Tricyclic Product at High Purity
[Image: see text] Terpenes are the largest class of natural products and are attractive targets in the fuel, fragrance, pharmaceutical, and flavor industries. Harvesting terpenes from natural sources is environmentally intensive and often gives low yields and purities, requiring further downstream processing. Engineered terpene synthases (TSs) offer a solution to these problems, but the low sequence identity and high promiscuity among TSs are major challenges for targeted engineering. Rational design of TSs requires identification of key structural and chemical motifs that steer product outcomes. Producing the sesquiterpenoid 10-epi-cubebol from farnesyl pyrophosphate (FPP) requires many steps and some of Nature’s most difficult chemistry. 10-epi-Cubebol synthase from Sorangium cellulosum (ScCubS) guides a highly reactive carbocationic substrate through this pathway, preventing early quenching and ensuring correct stereochemistry at every stage. The cyclizations carried out by ScCubS potentially represent significant evolutionary expansions in the chemical space accessible by TSs. Here, we present the high-resolution crystal structure of ScCubS in complex with both a trinuclear magnesium cluster and pyrophosphate. Computational modeling, experiment, and bioinformatic analysis identified residues important in steering the reaction chemistry. We show that S206 is crucial in 10-epi-cubebol synthesis by enlisting the nearby F211 to shape the active site contour and prevent the formation of early escape cadalane products. We also show that N327 and F104 control the distribution between several early-stage cations and whether the final product is derived from the germacrane, cadalane, or cubebane hydrocarbon scaffold. Using these insights, we reengineered ScCubS so that its main product was germacradien-4-ol, which derives from the germacrane, rather than the cubebane, scaffold. Our work emphasizes that mechanistic understanding of cation stabilization in TSs can be used to guide catalytic outcomes
Rewriting the Script: The Story of Vitamin C and the Epigenome
Vitamin C is a vital micronutrient in the maintenance of numerous cellular functions and the development of mammalian systems. Vitamin C predominantly exists physiologically as the ascorbate anion, an antioxidant classically linked to the prevention of scurvy. Current research has shown that ascorbate plays an additional role critical in DNA demethylation by acting as a cofactor for the ten-eleven translocation (TET) family of methylcytosine dioxygenase enzymes. TET enzymes hydroxylate 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), an epigenetic marker whose further processing results in cleavage of the methylated cytosine and subsequent repair via the base excision repair pathway, resulting in completion of active DNA demethylation. Recent work has also speculated ascorbate’s role in mediating histone demethylation dynamics via Jumonji C domain (JmjC) demethylase enzymes belonging to the same enzyme family as TET dioxygenases. Although these roles in demethylation are of principal importance, the need for ascorbate initially evolved in early photosynthetic eukaryotes who required a reducing agent to protect themselves from photodamage generated by the chloroplast, a role that ultimately affected the evolutionary paths of insects and herbivorous animals. Altogether, the wide-reaching functions of ascorbate play a critical role in the maintenance of mammalian demethylation dynamics and organismal development
Vpliv učenja in obsega žoge na spremembo hitrosti rokometnega strela
Thioredoxin, involved in numerous
redox pathways, is maintained
in the dithiol state by the nicotinamide adenine dinucleotide phosphate-dependent
flavoprotein thioredoxin reductase (TrxR). Here, TrxR from <i>Lactococcus lactis</i> is compared with the well-characterized
TrxR from <i>Escherichia coli</i>. The two enzymes belong
to the same class of low-molecular weight thioredoxin reductases and
display similar <i>k</i><sub>cat</sub> values (∼25
s<sup>–1</sup>) with their cognate thioredoxin. Remarkably,
however, the <i>L. lactis</i> enzyme is inactivated by visible
light and furthermore reduces molecular oxygen 10 times faster than <i>E. coli</i> TrxR. The rate of light inactivation under standardized
conditions (λ<sub>max</sub> = 460 nm and 4 °C) was reduced
at lowered oxygen concentrations and in the presence of iodide. Inactivation
was accompanied by a distinct spectral shift of the flavin adenine
dinucleotide (FAD) that remained firmly bound. High-resolution mass
spectrometric analysis of heat-extracted FAD from light-damaged TrxR
revealed a mass increment of 13.979 Da, relative to that of unmodified
FAD, corresponding to the addition of one oxygen atom and the loss
of two hydrogen atoms. Tandem mass spectrometry confined the increase
in mass of the isoalloxazine ring, and the extracted modified cofactor
reacted with dinitrophenyl hydrazine, indicating the presence of an
aldehyde. We hypothesize that a methyl group of FAD is oxidized to
a formyl group. The significance of this not previously reported oxidation
and the exceptionally high rate of oxygen reduction are discussed
in relation to other flavin modifications and the possible occurrence
of enzymes with similar properties