directed mutagenesis
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2022 ◽  
Vol 13 (1) ◽  
Author(s):  
Diane T. Takahashi ◽  
Danièle Gadelle ◽  
Keli Agama ◽  
Evgeny Kiselev ◽  
Hongliang Zhang ◽  
...  

AbstractEukaryotic topoisomerases I (TOP1) are ubiquitous enzymes removing DNA torsional stress. However, there is little data concerning the three-dimensional structure of TOP1 in the absence of DNA, nor how the DNA molecule can enter/exit its closed conformation. Here, we solved the structure of thermostable archaeal Caldiarchaeum subterraneum CsTOP1 in an apo-form. The enzyme displays an open conformation resulting from one substantial rotation between the capping (CAP) and the catalytic (CAT) modules. The junction between these two modules is a five-residue loop, the hinge, whose flexibility permits the opening/closing of the enzyme and the entry of DNA. We identified a highly conserved tyrosine near the hinge as mediating the transition from the open to closed conformation upon DNA binding. Directed mutagenesis confirmed the importance of the hinge flexibility, and linked the enzyme dynamics with sensitivity to camptothecin, a TOP1 inhibitor targeting the TOP1 enzyme catalytic site in the closed conformation.


ACS Omega ◽  
2021 ◽  
Author(s):  
Sreejana Ray ◽  
Desiree Tillo ◽  
Nima Assad ◽  
Aniekanabasi Ufot ◽  
Aleksey Porollo ◽  
...  

PLoS Biology ◽  
2021 ◽  
Vol 19 (12) ◽  
pp. e3001498
Author(s):  
Haiyang Wu ◽  
Emmanuelle H. Crost ◽  
C David Owen ◽  
Wouter van Bakel ◽  
Ana Martínez Gascueña ◽  
...  

The human gut symbiont Ruminococcus gnavus displays strain-specific repertoires of glycoside hydrolases (GHs) contributing to its spatial location in the gut. Sequence similarity network analysis identified strain-specific differences in blood-group endo-β-1,4-galactosidase belonging to the GH98 family. We determined the substrate and linkage specificities of GH98 from R. gnavus ATCC 29149, RgGH98, against a range of defined oligosaccharides and glycoconjugates including mucin. We showed by HPAEC-PAD and LC-FD-MS/MS that RgGH98 is specific for blood group A tetrasaccharide type II (BgA II). Isothermal titration calorimetry (ITC) and saturation transfer difference (STD) NMR confirmed RgGH98 affinity for blood group A over blood group B and H antigens. The molecular basis of RgGH98 strict specificity was further investigated using a combination of glycan microarrays, site-directed mutagenesis, and X-ray crystallography. The crystal structures of RgGH98 in complex with BgA trisaccharide (BgAtri) and of RgGH98 E411A with BgA II revealed a dedicated hydrogen network of residues, which were shown by site-directed mutagenesis to be critical to the recognition of the BgA epitope. We demonstrated experimentally that RgGH98 is part of an operon of 10 genes that is overexpresssed in vitro when R. gnavus ATCC 29149 is grown on mucin as sole carbon source as shown by RNAseq analysis and RT-qPCR confirmed RgGH98 expression on BgA II growth. Using MALDI-ToF MS, we showed that RgGH98 releases BgAtri from mucin and that pretreatment of mucin with RgGH98 confered R. gnavus E1 the ability to grow, by enabling the E1 strain to metabolise BgAtri and access the underlying mucin glycan chain. These data further support that the GH repertoire of R. gnavus strains enable them to colonise different nutritional niches in the human gut and has potential applications in diagnostic and therapeutics against infection.


eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Tümay Capraz ◽  
Nikolaus Ferdinand Kienzl ◽  
Elisabeth Laurent ◽  
Jan W Perthold ◽  
Esther Föderl-Höbenreich ◽  
...  

Infection and viral entry of SARS-CoV-2 crucially depends on the binding of its Spike protein to angiotensin converting enzyme 2 (ACE2) presented on host cells. Glycosylation of both proteins is critical for this interaction. Recombinant soluble human ACE2 can neutralize SARS-CoV-2 and is currently undergoing clinical tests for the treatment of COVID-19. We used 3D structural models and molecular dynamics simulations to define the ACE2 N-glycans that critically influence Spike-ACE2 complex formation. Engineering of ACE2 N-glycosylation by site-directed mutagenesis or glycosidase treatment resulted in enhanced binding affinities and improved virus neutralization without notable deleterious effects on the structural stability and catalytic activity of the protein. Importantly, simultaneous removal of all accessible N-glycans from recombinant soluble human ACE2 yields a superior SARS-CoV-2 decoy receptor with promise as effective treatment for COVID-19 patients.


2021 ◽  
Vol 2 (4) ◽  
pp. 96-105
Author(s):  
Raghad Abed ◽  
Yusra Al-Najjar

An exceptional branch of data that requires huge databases has been shown lately from genome sequencing projects which is a field that employs computational approaches to answer biological questions. With this huge sequence of information that is available for researchers, bioinformatics plays a big role in studying basic medical-biological problems. The challenge that faces bioinformatical scientists is to help in discovering genes and designing molecular models, site-directed mutagenesis, and other experiments that reveal the unknown relationships concerning the structure and function of genes and proteins. This become a big challenge especially with the huge amount of data that is generated using the human genome and other systematic sequencing efforts up till now. Bioinformatics solves biological problems depending on available data. It is concerned with creating databases and predicting the outcome of lab experiments.


2021 ◽  
Vol 22 (24) ◽  
pp. 13206
Author(s):  
Gabino Francisco Sánchez-Pérez ◽  
María Ángeles Pajares

Catalytic MATα1 subunits associate into kinetically distinct homo-dimers (MAT III) and homo-tetramers (MAT I) that synthesize S-adenosylmethionine in the adult liver. Pathological reductions in S-adenosylmethionine levels correlate with MAT III accumulation; thus, it is important to know the determinants of dimer–dimer associations. Here, polar interactions (<3.5 Å) at the rat MAT I dimer–dimer interface were disrupted by site-directed mutagenesis. Heterologous expression rendered decreased soluble mutant MATα1 levels that appeared mostly as dimers. Substitutions at the B1–B2 or B3–C1 β-strand loops, or changes in charge on helix α2 located behind, induced either MAT III or MAT I accumulation. Notably, double mutants combining neutral changes on helix α2 with substitutions at either β-strand loop further increased MAT III content. Mutations had negligible impact on secondary or tertiary protein structure, but induced changes of 5–10 °C in thermal stability. All mutants preserved tripolyphosphatase activity, although AdoMet synthesis was only detected in single mutants. Kinetic parameters were altered in all purified proteins, their AdoMet synthesis Vmax and methionine affinities correlating with the association state induced by the corresponding mutations. In conclusion, polar interactions control MATα1 tetramerization and kinetics, diverse effects being induced by changes on opposite β-sheet loops putatively leading to subtle variations in central domain β-sheet orientation.


2021 ◽  
Author(s):  
◽  
Kelsi Hall

<p>Bacterial nitroreductases are flavoenzymes able to catalyse the reduction of nitroaromatic compounds. The research presented in this thesis focused on NfsA_Ec, a nitroreductase from E. coli. NfsA_Ec is a promiscuous enzyme that can reduce a wide range of nitroaromatic antibiotics and prodrugs. This research sought to use NfsA_Ec as a model to improve our understanding of directed evolution, and also to identify NfsA_Ec variants exhibiting improved activation with a range of nil-bystander prodrugs for use in a targeted cell ablation system in zebrafish.  There is a substantial gap between the levels of enzyme activity that nature can achieve and those that scientists can evolve in the lab. This suggests that conventional directed evolution techniques involving incremental improvements in enzyme activity may frequently fail to ascend even local fitness maxima. We sought to contrast such approaches with simultaneous site-directed mutagenesis, employing a library of 252 million unique nfsA variants. To determine whether two superior NfsA_Ec variants recovered from this library could have been identified using a conventional stepwise approach we generated all possible intermediates of these two enzyme variants and recreated the most logical evolutionary trajectory for each enzyme variant. This revealed that a stepwise mutagenesis approach could indeed have yielded both of these variants, but also that very few evolutionary trajectories were accessible due to complex epistatic interactions between substitutions in these enzymes. Moreover, many conventional stepwise mutagenesis approaches such as iterative saturation mutagenesis would have failed to identify key substitutions in these variants. We also investigated the “black-box” effect of directed evolution, using NfsA_Ec and a panel of nitroaromatic compounds to model the off-target effects an evolved enzyme can have within an existing metabolic network. We found that selection for improved niclosamide and chloramphenicol detoxification also improved activity with some structurally distinct prodrugs, but not others. Using a dual positive-negative selection, we recovered NfsA_Ec variants that were more specialised for their primary activities, however this came at a cost in terms of overall activity levels.  The simultaneous site-directed nfsA_Ec mutagenesis library also had practical applications, enabling recovery of NfsA_Ec variants for targeted cell ablation in zebrafish models. These models involve the selective ablation of nitroreductase expressing cells without harming adjacent cells, to mimic a degenerative disease. Several NfsA_Ec variants were identified which were highly active with the nil-bystander prodrugs metronidazole, tinidazole, RB6145 and misonidazole when expressed in E. coli. However, these NfsA_Ec variants had inconsistent activities in our eukaryotic cell model (HEK-293). To expand the utility of the core ablation system, we sought to identify pairs of nitroreductases with non-overlapping prodrug specificities, suitable for use in a multiplex cell ablation system. Using a dual positive-negative selection, we recovered several NfsA_Ec variants that exhibited preferential nitrofurazone activation over metronidazole. Our lead variants for both applications are currently being trialed in zebrafish for their utility in generating degenerative disease models.</p>


2021 ◽  
Author(s):  
◽  
Kelsi Hall

<p>Bacterial nitroreductases are flavoenzymes able to catalyse the reduction of nitroaromatic compounds. The research presented in this thesis focused on NfsA_Ec, a nitroreductase from E. coli. NfsA_Ec is a promiscuous enzyme that can reduce a wide range of nitroaromatic antibiotics and prodrugs. This research sought to use NfsA_Ec as a model to improve our understanding of directed evolution, and also to identify NfsA_Ec variants exhibiting improved activation with a range of nil-bystander prodrugs for use in a targeted cell ablation system in zebrafish.  There is a substantial gap between the levels of enzyme activity that nature can achieve and those that scientists can evolve in the lab. This suggests that conventional directed evolution techniques involving incremental improvements in enzyme activity may frequently fail to ascend even local fitness maxima. We sought to contrast such approaches with simultaneous site-directed mutagenesis, employing a library of 252 million unique nfsA variants. To determine whether two superior NfsA_Ec variants recovered from this library could have been identified using a conventional stepwise approach we generated all possible intermediates of these two enzyme variants and recreated the most logical evolutionary trajectory for each enzyme variant. This revealed that a stepwise mutagenesis approach could indeed have yielded both of these variants, but also that very few evolutionary trajectories were accessible due to complex epistatic interactions between substitutions in these enzymes. Moreover, many conventional stepwise mutagenesis approaches such as iterative saturation mutagenesis would have failed to identify key substitutions in these variants. We also investigated the “black-box” effect of directed evolution, using NfsA_Ec and a panel of nitroaromatic compounds to model the off-target effects an evolved enzyme can have within an existing metabolic network. We found that selection for improved niclosamide and chloramphenicol detoxification also improved activity with some structurally distinct prodrugs, but not others. Using a dual positive-negative selection, we recovered NfsA_Ec variants that were more specialised for their primary activities, however this came at a cost in terms of overall activity levels.  The simultaneous site-directed nfsA_Ec mutagenesis library also had practical applications, enabling recovery of NfsA_Ec variants for targeted cell ablation in zebrafish models. These models involve the selective ablation of nitroreductase expressing cells without harming adjacent cells, to mimic a degenerative disease. Several NfsA_Ec variants were identified which were highly active with the nil-bystander prodrugs metronidazole, tinidazole, RB6145 and misonidazole when expressed in E. coli. However, these NfsA_Ec variants had inconsistent activities in our eukaryotic cell model (HEK-293). To expand the utility of the core ablation system, we sought to identify pairs of nitroreductases with non-overlapping prodrug specificities, suitable for use in a multiplex cell ablation system. Using a dual positive-negative selection, we recovered several NfsA_Ec variants that exhibited preferential nitrofurazone activation over metronidazole. Our lead variants for both applications are currently being trialed in zebrafish for their utility in generating degenerative disease models.</p>


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