scholarly journals Single point mutations reveal amino acid residues important for Chromobacterium violaceum transaminase activity in the production of unnatural amino acids

2018 ◽  
Vol 8 (1) ◽  
Author(s):  
Sarah A. Almahboub ◽  
Tanja Narancic ◽  
Darren Fayne ◽  
Kevin E. O’Connor
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Unnatural Amino Acids ◽  
Single Point ◽  
Point Mutations ◽  
Chromobacterium Violaceum ◽  
Amino Acid Residues ◽  
Transaminase Activity ◽  
Single Point Mutations

scholarly journals The Effect of a Peptide Substrate Containing an Unnatural Branched Amino Acid on Chymotrypsin Activity

Processes ◽  
2021 ◽  
Vol 9 (2) ◽  
pp. 242
Author(s):  
Yuuki Yamawaki ◽  
Tomoki Yufu ◽  
Tamaki Kato
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Substrate Specificity ◽  
Unnatural Amino Acids ◽  
Side Chain ◽  
Low Molecular Weight ◽  
Amino Acid Residues ◽  
Phase Synthesis ◽  
Reaction Velocity ◽  
Natural Amino Acids

7-Amino-4-methylcoumarin (AMC) is a low molecular weight fluorescent probe that can be attached to a peptide to enable the detection of specific proteases, such as chymotrypsin, expressed in certain diseases. Because this detection depends on the specificity of the protease toward the peptidyl AMC, the development of specific substrates is required. To investigate the specificity of chymotrypsin, peptidyl AMC compounds incorporating four different amino acid residues were prepared by liquid-phase synthesis. Two unnatural amino acids, 2-amino-4-ethylhexanoic acid (AEH) and cyclohexylalanine (Cha), were used to investigate the substrate specificity as these amino acids have structures different from natural amino acids. AEH was synthesized using diethyl acetamidemalonate as a starting material. The substrate containing Cha had high hydrophobicity and showed a high reaction velocity with chymotrypsin. Although the AEH substrate with a branched side chain had high hydrophobicity, it showed a low reaction velocity. The substrate containing the aromatic amino acid phenylalanine was less hydrophobic than the Cha and AEH substrates, but chymotrypsin showed the highest specificity for this compound. These results demonstrated that the substrate specificity of chymotrypsin is not only affected by the hydrophobicity and aromaticity, but also by the structural expanse of amino acid residues in the substrate.

scholarly journals Visualizing Deep Mutational Scan Data

2018 ◽  
Author(s):  
C. K. Sruthi ◽  
Hemalatha Balaram ◽  
Meher K. Prakash
Keyword(s):  
Amino Acid ◽  
Single Point ◽  
Point Mutations ◽  
Random Mutagenesis ◽  
Opportunity To Learn ◽  
Single Point Mutations ◽  
The Rich ◽  
Amino Acid Mutations ◽  
Rich Data ◽  
And Function

AbstractSite-directed and random mutagenesis are biochemical tools to obtain insights into the structure and function of proteins. Recent advances such as deep mutational scan have allowed a complete scan of all the amino acid positions in a protein with each of the 19 possible alternatives. Mapping out the phenotypic consequences of thousands of single point mutations in the same protein is now possible. Visualizing and analysing the rich data offers an opportunity to learn more about the effects of mutations, for a better understanding and engineering of proteins. This work focuses on such visualization analyses applied to the mutational data of TEM-1 β-lactamase. The data is examined in the light of the expected biochemical effects of single point mutations, with the goal of reinforcing or retraining the intuitions. Individual attributes of the amino acid mutations such as the solvent accessible area, charge type change, and distance from the catalytic center capture most of the relevant functional effects. Visualizing the data suggests how combinations of these attributes can be used for a better classification of the effects of mutations, when independently they do not offer a high predictability.

scholarly journals Two single-point mutations shift the ligand selectivity of a pheromone receptor between two closely related moth species

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Ke Yang ◽  
Ling-Qiao Huang ◽  
Chao Ning ◽  
Chen-Zhu Wang
Keyword(s):  
Amino Acid ◽  
Single Point ◽  
Point Mutations ◽  
Functional Difference ◽  
Amino Acid Residues ◽  
Helicoverpa Assulta ◽  
Ligand Selectivity ◽  
Olfactory Systems ◽  
Moth Species ◽  
Species Specific

Male moths possess highly sensitive and selective olfactory systems that detect sex pheromones produced by their females. Pheromone receptors (PRs) play a key role in this process. The PR HassOr14b is found to be tuned to (Z)−9-hexadecenal, the major sex-pheromone component, in Helicoverpa assulta. HassOr14b is co-localized with HassOr6 or HassOr16 in two olfactory sensory neurons within the same sensilla. As HarmOr14b, the ortholog of HassOr14b in the closely related species Helicoverpa armigera, is tuned to another chemical (Z)−9-tetradecenal, we study the amino acid residues that determine their ligand selectivity. Two amino acids located in the transmembrane domains F232I and T355I together determine the functional difference between the two orthologs. We conclude that species-specific changes in the tuning specificity of the PRs in the two Helicoverpa moth species could be achieved with just a few amino acid substitutions, which provides new insights into the evolution of closely related moth species.

Cupin Variants as Macromolecular Ligand Library for Stereoselective Michael Addition of Nitroalkanes

2019 ◽  
Author(s):  
Nobutaka Fujieda ◽  
Miho Yuasa ◽  
Yosuke Nishikawa ◽  
Genji Kurisu ◽  
Shinobu Itoh ◽  
...  
Keyword(s):  
Michael Addition ◽  
In Silico ◽  
Addition Reaction ◽  
Single Point ◽  
Point Mutations ◽  
Unsaturated Ketone ◽  
Michael Addition Reaction ◽  
Beta Barrel ◽  
Cupin Superfamily ◽  
Single Point Mutations

Cupin superfamily proteins (TM1459) work as a macromolecular ligand framework with a double-stranded beta-barrel structure ligating to a Cu ion through histidine side chains. Variegating the first coordination sphere of TM1459 revealed that H52A and H54A/H58A mutants effectively catalyzed the diastereo- and enantio-selective Michael addition reaction of nitroalkanes to an α,β-unsaturated ketone. Moreover, in silico substrate docking signified C106N and F104W single-point mutations, which inverted the diastereoselectivity of H52A and further improved the stereoselectivity of H54A/H58A, respectively.

Single-Point Mutations in Qβ Virus-like Particles Change Binding to Cells

2021 ◽  
Author(s):  
Marisa L. Martino ◽  
Stephen N. Crooke ◽  
Marianne Manchester ◽  
M.G. Finn
Keyword(s):  
Single Point ◽  
Point Mutations ◽  
Virus Like Particles ◽  
Single Point Mutations

scholarly journals Transferability of N-terminal mutations of pyrrolysyl-tRNA synthetase in one species to that in another species on unnatural amino acid incorporation efficiency

Amino Acids ◽  
2020 ◽  
Author(s):  
Thomas L. Williams ◽  
Debra J. Iskandar ◽  
Alexander R. Nödling ◽  
Yurong Tan ◽  
Louis Y. P. Luk ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Genetic Code ◽  
Unnatural Amino Acids ◽  
Trna Synthetase ◽  
Unnatural Amino Acid ◽  
Amino Acid Incorporation ◽  
Acid Incorporation ◽  
Genetic Code Expansion ◽  
Incorporation Efficiency

AbstractGenetic code expansion is a powerful technique for site-specific incorporation of an unnatural amino acid into a protein of interest. This technique relies on an orthogonal aminoacyl-tRNA synthetase/tRNA pair and has enabled incorporation of over 100 different unnatural amino acids into ribosomally synthesized proteins in cells. Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA from Methanosarcina species are arguably the most widely used orthogonal pair. Here, we investigated whether beneficial effect in unnatural amino acid incorporation caused by N-terminal mutations in PylRS of one species is transferable to PylRS of another species. It was shown that conserved mutations on the N-terminal domain of MmPylRS improved the unnatural amino acid incorporation efficiency up to five folds. As MbPylRS shares high sequence identity to MmPylRS, and the two homologs are often used interchangeably, we examined incorporation of five unnatural amino acids by four MbPylRS variants at two temperatures. Our results indicate that the beneficial N-terminal mutations in MmPylRS did not improve unnatural amino acid incorporation efficiency by MbPylRS. Knowledge from this work contributes to our understanding of PylRS homologs which are needed to improve the technique of genetic code expansion in the future.

MinD directly interacting with FtsZ at the H10 helix suggests a model for robust activation of MinC to destabilize FtsZ polymers

2017 ◽  
Vol 474 (18) ◽  
pp. 3189-3205 ◽  
Author(s):  
Ashoka Chary Taviti ◽  
Tushar Kant Beuria
Keyword(s):  
Cell Division ◽  
Single Point ◽  
Point Mutations ◽  
Direct Interaction ◽  
Ring Formation ◽  
Z Ring ◽  
Single Point Mutations ◽  
Biochemical Analyses ◽  
Ftsz Assembly ◽  
The Mind

Cell division in bacteria is a highly controlled and regulated process. FtsZ, a bacterial cytoskeletal protein, forms a ring-like structure known as the Z-ring and recruits more than a dozen other cell division proteins. The Min system oscillates between the poles and inhibits the Z-ring formation at the poles by perturbing FtsZ assembly. This leads to an increase in the FtsZ concentration at the mid-cell and helps in Z-ring positioning. MinC, the effector protein, interferes with Z-ring formation through two different mechanisms mediated by its two domains with the help of MinD. However, the mechanism by which MinD triggers MinC activity is not yet known. We showed that MinD directly interacts with FtsZ with an affinity stronger than the reported MinC–FtsZ interaction. We determined the MinD-binding site of FtsZ using computational, mutational and biochemical analyses. Our study showed that MinD binds to the H10 helix of FtsZ. Single-point mutations at the charged residues in the H10 helix resulted in a decrease in the FtsZ affinity towards MinD. Based on our findings, we propose a novel model for MinCD–FtsZ interaction, where MinD through its direct interaction with FtsZ would trigger MinC activity to inhibit FtsZ functions.

scholarly journals Identification of separate domains in the adenovirus E1A gene for immortalization activity and the activation of virus early genes.

1986 ◽  
Vol 6 (10) ◽  
pp. 3470-3480 ◽  
Author(s):  
E Moran ◽  
B Zerler ◽  
T M Harrison ◽  
M B Mathews
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Point Mutations ◽  
Apparent Molecular Weight ◽  
Amino Acid Position ◽  
Cysteine Residue ◽  
Transformation Function ◽  
Single Amino Acid ◽  
Amino Acid Substitutions ◽  
E1a Gene

The transformation and early adenovirus gene transactivation functions of the E1A region were analyzed with deletion and point mutations. Deletion of amino acids from position 86 through 120 had little effect on the lytic or transforming functions of the E1A products, while deletion of amino acids from position 121 through 150 significantly impaired both functions. The sensitivity of the transformation function to alterations in the region from amino acid position 121 to 150 was further indicated by the impairment of transforming activity resulting from single amino acid substitutions at positions 124 and 135. Interestingly, conversion of a cysteine residue at position 124 to glycine severely impaired the transformation function without affecting the early adenovirus gene activating functions. Single amino acid substitutions in a different region of the E1A gene had the converse effect. All the mutants produced polypeptides of sufficient stability to be detected by Western immunoblot analysis. The single amino acid substitutions at positions 124 and 135, although impairing the transformation functions, did not detectably alter the formation of the higher-apparent-molecular-weight forms of the E1A products.

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