scholarly journals Using structurally fungible biosensors to evolve improved alkaloid biosyntheses

Author(s):  
Simon d'Oelsnitz ◽  
Wantae Kim ◽  
Nathaniel Burkholder ◽  
Kamyab Javanmardi ◽  
Ross Thyer ◽  
...  

Abstract A key bottleneck in the microbial production of therapeutic plant metabolites is identifying enzymes that can greatly improve yield. The facile identification of genetically encoded biosensors can overcome this limitation and become part of a general method for engineering scaled production. We have developed a unique combined screening and selection approach that quickly refines the affinities and specificities of generalist transcription factors, and using RamR as a starting point we evolve highly specific (>100-fold preference) and sensitive (EC50 <30 μM) biosensors for the alkaloids tetrahydropapaverine, papaverine, glaucine, rotundine, and noscapine. High resolution structures reveal multiple evolutionary avenues for the fungible effector binding site, and the creation of new pockets for different chemical moieties. These sensors further enabled the evolution of a streamlined pathway for tetrahydropapaverine, an immediate precursor to four modern pharmaceuticals, collapsing multiple methylation steps into a single evolved enzyme. Our methods for evolving biosensors now enable the rapid engineering of pathways for therapeutic alkaloids.

2021 ◽  
Author(s):  
Simon d'Oelsnitz ◽  
Wantae Kim ◽  
Nathaniel T Burkholder ◽  
Kamyab javanmardi ◽  
Ross Thyer ◽  
...  

A key bottleneck in the microbial production of therapeutic plant metabolites is identifying enzymes that can greatly improve yield. The facile identification of genetically encoded biosensors can overcome this limitation and become part of a general method for engineering scaled production. We have developed a unique combined screening and selection approach that quickly refines the affinities and specificities of generalist transcription factors, and using RamR as a starting point we evolve highly specific (>100-fold preference) and sensitive (EC50 <30 μM) biosensors for the alkaloids tetrahydropapaverine, papaverine, glaucine, rotundine, and noscapine. High resolution structures reveal multiple evolutionary avenues for the fungible effector binding site, and the creation of new pockets for different chemical moieties. These sensors further enabled the evolution of a streamlined pathway for tetrahydropapaverine, an immediate precursor to four modern pharmaceuticals, collapsing multiple methylation steps into a single evolved enzyme. Our methods for evolving biosensors now enable the rapid engineering of pathways for therapeutic alkaloids.


Trictrac ◽  
2016 ◽  
Vol 9 ◽  
Author(s):  
Liliana Danciu ◽  
Petru Adrian Danciu

The axes of the creation and birth of the imaginary as a mythical language. Our research follows the relationships of the concepts that are taking into account creation on the double axis of verticality and horizontality. We highlight those symbolic elements which would later constitute the mythical language about the sacred space-temporality. Inside this space-temporality a rich spectrum of mythical images develops; images capable of explaining the relationships of the creation plans. Without a religious perception of the temporality, the conceptualization of the axis would remain a philosophical approach. Through our point of view, the two are born simultaneously. Thanks to them, creation can be imagined. The first “frozen” formula of the mystical human spirit can be thought, brought to a palpable reality, expressed in an oral and then a written form. Studied together, temporality (sacred or not) and space are permanently imagined together. For example, a loss of mundane temporality in the secret ecstasy that offers to the soul an ascending direction does not mean getting out of universal temporality, but of its mundane section. In the sacred space the soul relates to time. Even the gods are submitted by the sacred, Aeon sometimes being synonymous to destiny. The universal creator seems to evade every touch, but not consistently, only when he avoids the descent into its created worlds. In sacredness, time and space seem or become confused, both expressing the same reality, by the immediate swing from thinking to deed. The mythical imagery conceives the displacement in the primary space-temporality by the spoken word. So, for something to appear and live, the spoken word is required. Even the divine dream appears as a pre-word of a creator’s thought. The thought follows the spoken word, the spoken word follows the gestures which finally indicate the meanings of the creative act, controlling the rhythm of the creation days. These three will later be adapted through imitation in rite. We are now situated at the limit of the physical world, a real challenge for the mythical imagery. The general feature of the mythical expression on the creation of the material world is the state of the divinity’s exhaustion, most often conceptualized by sacrifice or divine fatigue. The world geography identifies with the anatomy of a self-gutted god. Practically, material creation is most likely the complete revelation of God’s body autopsy. As each body decomposes, everything in it is an illusion. An axial approach of the phenomenon exists in all religious systems. The created element’s origin is exterior, with or without a pre-existing matter, by a god’s sacrifice or only because it has to be that way. This is the starting point of the discussion on the symbolism of axiality as a reason for the constitution of the language of creation, capable of retelling the imaginary construction of myth in an oral and then written form.


2020 ◽  
Vol 16 (6) ◽  
pp. 784-795
Author(s):  
Krisnna M.A. Alves ◽  
Fábio José Bonfim Cardoso ◽  
Kathia M. Honorio ◽  
Fábio A. de Molfetta

Background:: Leishmaniosis is a neglected tropical disease and glyceraldehyde 3- phosphate dehydrogenase (GAPDH) is a key enzyme in the design of new drugs to fight this disease. Objective:: The present study aimed to evaluate potential inhibitors of GAPDH enzyme found in Leishmania mexicana (L. mexicana). Methods: A search for novel antileishmanial molecules was carried out based on similarities from the pharmacophoric point of view related to the binding site of the crystallographic enzyme using the ZINCPharmer server. The molecules selected in this screening were subjected to molecular docking and molecular dynamics simulations. Results:: Consensual analysis of the docking energy values was performed, resulting in the selection of ten compounds. These ligand-receptor complexes were visually inspected in order to analyze the main interactions and subjected to toxicophoric evaluation, culminating in the selection of three compounds, which were subsequently submitted to molecular dynamics simulations. The docking results showed that the selected compounds interacted with GAPDH from L. mexicana, especially by hydrogen bonds with Cys166, Arg249, His194, Thr167, and Thr226. From the results obtained from molecular dynamics, it was observed that one of the loop regions, corresponding to the residues 195-222, can be related to the fitting of the substrate at the binding site, assisting in the positioning and the molecular recognition via residues responsible for the catalytic activity. Conclusion:: he use of molecular modeling techniques enabled the identification of promising compounds as inhibitors of the GAPDH enzyme from L. mexicana, and the results obtained here can serve as a starting point to design new and more effective compounds than those currently available.


2008 ◽  
Vol 7 (1) ◽  
pp. 64-76
Author(s):  
Andy Stirrup

This paper considers an implicit trend in youth ministry to present Jesus as the archetypal superhero and asks if this is a valid and a helpful approach. The paper examines the relationship between the biblical category of hero and the contemporary notion of superhero and a broader appreciation of the use of myth for communicating Christian apologetics as seen in Lewis and Tolkien. The starting point for the paper is that an arguable starting point for the creation of Superman is in the epic character of Hercules and the biblical hero Samson. Through an examination of biblical and other Near East material the paper calls for a deeper and more nuanced appreciation of the relevance of modern western myth in the task of communicating theological narratives and concepts.


2020 ◽  
Vol 22 (19) ◽  
pp. 10853-10862
Author(s):  
Kenta Mizuse ◽  
Naoya Sakamoto ◽  
Romu Fujimoto ◽  
Yasuhiro Ohshima

High-resolution molecular movies of direction-controlled rotational wave packets are reported, providing insights into the creation process and detailed dynamics of wave packets.


2011 ◽  
Vol 39 (5) ◽  
pp. 1353-1358 ◽  
Author(s):  
Simon Newstead

One of the principal aims of modern drug design is the targeted delivery of drugs within the body, such as to the central nervous system, combined with their exclusion from the liver and kidneys, which break down foreign molecules and subsequently eliminate them. Many of the commonly prescribed drugs are transported into cells and across the plasma membrane via endogenous membrane transporters, whose principal roles are the uptake of essential nutrients for metabolism. In many cases, such drug transport is serendipitous as they are simply mistaken as ‘natural’ compounds. Many of these transporters could, however, be targeted more efficiently, improving drug absorption, distribution and retention. The molecular details of these drug–transporter interactions, however, are at best poorly understood, in large part through the absence of any high-resolution structural information. To address this issue, we recently determined the structure of a prokaryotic peptide transporter, PepTSo from Shewanella oneidensis, which shares a high degree of sequence similarity and functional characteristics with the human PepT1 and PepT2 proteins. PepT1 and PepT2 contribute significantly to the oral bioavailability and pharmacokinetic properties of a number of important drug families, including antibiotics, antivirals and anticancer agents. The crystal structure of PepTSo provides the first high-resolution model of a drug importer and provides the starting point for understanding drug and peptide transport within the human body.


1994 ◽  
Vol 14 (6) ◽  
pp. 4116-4125 ◽  
Author(s):  
M L Espinás ◽  
J Roux ◽  
J Ghysdael ◽  
R Pictet ◽  
T Grange

We have previously shown that two remote glucocorticoid-responsive units (GRUs) of the rat tyrosine aminotransferase (TAT) gene contain multiple binding sites for several transcription factor families, including the glucocorticoid receptor (GR). We report here the identification of two novel binding sites for members of the Ets family of transcription factors in one of these GRUs. One of these binding sites overlaps the major GR-binding site (GRBS), whereas the other is located in its vicinity. Inactivation of the latter binding site leads to a twofold reduction of the glucocorticoid response, whereas inactivation of the site overlapping the GRBS has no detectable effect. In vivo footprinting analysis reveals that the active site is occupied in a glucocorticoid-independent manner, in a TAT-expressing cell line, even though it is located at a position where there is a glucocorticoid-dependent alteration of the nucleosomal structure. This same site is not occupied in a cell line that does not express TAT but expresses Ets-related DNA-binding activities, suggesting the existence of an inhibitory effect of chromatin structure at a hierarchical level above the nucleosome. The inactive Ets-binding site that overlaps the GRBS is not occupied even in TAT-expressing cells. However, this same overlapping site can confer Ets-dependent stimulation of both basal and glucocorticoid-induced levels when it is isolated from the GRU and duplicated. Ets-1 expression in COS cells mimics the activity of the Ets-related activities present in hepatoma cells. These Ets-binding sites could participate in the integration of the glucocorticoid response of the TAT gene with signal transduction pathways triggered by other nonsteroidal extracellular stimuli.


2020 ◽  
Author(s):  
Daniel J. Goetschius ◽  
Samantha R. Hartmann ◽  
Lindsey J. Organtini ◽  
Heather Callaway ◽  
Kai Huang ◽  
...  

AbstractOverlap on the surface of parvovirus capsids between the antigenic epitope and the receptor binding site contributes to species jumping. Mab 14 strongly binds and neutralizes canine, but not feline parvovirus. The high resolution map of the canine parvovirus capsid complexed with Fab 14 was used to solve local structures of the Fab-bound and -unbound antigenic sites extracted from the same complex. The subsequent analysis includes a new method for using cryo EM to investigate complementarity of antibody binding.


IUCrJ ◽  
2019 ◽  
Vol 6 (2) ◽  
pp. 238-247 ◽  
Author(s):  
Jimi M. Alex ◽  
Martin L. Rennie ◽  
Sylvain Engilberge ◽  
Gábor Lehoczki ◽  
Hajdu Dorottya ◽  
...  

Synthetic macrocycles such as calixarenes and cucurbiturils are increasingly applied as mediators of protein assembly and crystallization. The macrocycle can facilitate assembly by providing a surface on which two or more proteins bind simultaneously. This work explores the capacity of the sulfonato-calix[n]arene (sclx n ) series to effect crystallization of PAF, a small, cationic antifungal protein. Co-crystallization with sclx4, sclx6 or sclx8 led to high-resolution crystal structures. In the absence of sclx n , diffraction-quality crystals of PAF were not obtained. Interestingly, all three sclx n were bound to a similar patch on PAF. The largest and most flexible variant, sclx8, yielded a dimer of PAF. Complex formation was evident in solution via NMR and ITC experiments, showing more pronounced effects with increasing macrocycle size. In agreement with the crystal structure, the ITC data suggested that sclx8 acts as a bidentate ligand. The contributions of calixarene size/conformation to protein recognition and assembly are discussed. Finally, it is suggested that the conserved binding site for anionic calixarenes implicates this region of PAF in membrane binding, which is a prerequisite for antifungal activity.


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