Formylglycine-generating enzymes for site-specific bioconjugation

2019 ◽  
Vol 400 (3) ◽  
pp. 289-297 ◽  
Author(s):  
Tobias Krüger ◽  
Thomas Dierks ◽  
Norbert Sewald
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Recombinant Proteins ◽  
The State ◽  
Protein Scaffolds ◽  
Site Specific ◽  
Catalytic Mechanisms ◽  
Consensus Motifs ◽  
State Of Research ◽  
Natural Amino Acids

Abstract Site-specific bioconjugation strategies offer many possibilities for directed protein modifications. Among the various enzyme-based conjugation protocols, formylglycine-generating enzymes allow to posttranslationally introduce the amino acid Cα-formylglycine (FGly) into recombinant proteins, starting from cysteine or serine residues within distinct consensus motifs. The aldehyde-bearing FGly-residue displays orthogonal reactivity to all other natural amino acids and can, therefore, be used for site-specific labeling reactions on protein scaffolds. In this review, the state of research on catalytic mechanisms and consensus motifs of different formylglycine-generating enzymes, as well as labeling strategies and applications of FGly-based bioconjugations are presented.

Engineering an acetyllysine reader with photocrosslinking amino acid for interactome profiling

2021 ◽  
Author(s):  
Babu Sudhamalla ◽  
Anirban Roy ◽  
Soumen Barman ◽  
Jyotirmayee Padhan
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Protein Interactions ◽  
Unnatural Amino Acids ◽  
Protein Protein Interactions ◽  
Site Specific ◽  
Powerful Approach

The site-specific installation of light-activable crosslinker unnatural amino acids offers a powerful approach to trap transient protein-protein interactions both in vitro and in vivo. Herein, we engineer a bromodomain to...

scholarly journals Structural basis for divergent and convergent evolution of catalytic machineries in plant aromatic amino acid decarboxylase proteins

2020 ◽  
Vol 117 (20) ◽  
pp. 10806-10817 ◽  
Author(s):  
Michael P. Torrens-Spence ◽  
Ying-Chih Chiang ◽  
Tyler Smith ◽  
Maria A. Vicent ◽  
Yi Wang ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Convergent Evolution ◽  
Structural Features ◽  
Divergent Evolution ◽  
Structural Basis ◽  
Acid Decarboxylase ◽  
Substrate Selectivity ◽  
Amino Acid Decarboxylase ◽  
Catalytic Mechanisms

Radiation of the plant pyridoxal 5′-phosphate (PLP)-dependent aromatic l-amino acid decarboxylase (AAAD) family has yielded an array of paralogous enzymes exhibiting divergent substrate preferences and catalytic mechanisms. Plant AAADs catalyze either the decarboxylation or decarboxylation-dependent oxidative deamination of aromatic l-amino acids to produce aromatic monoamines or aromatic acetaldehydes, respectively. These compounds serve as key precursors for the biosynthesis of several important classes of plant natural products, including indole alkaloids, benzylisoquinoline alkaloids, hydroxycinnamic acid amides, phenylacetaldehyde-derived floral volatiles, and tyrosol derivatives. Here, we present the crystal structures of four functionally distinct plant AAAD paralogs. Through structural and functional analyses, we identify variable structural features of the substrate-binding pocket that underlie the divergent evolution of substrate selectivity toward indole, phenyl, or hydroxyphenyl amino acids in plant AAADs. Moreover, we describe two mechanistic classes of independently arising mutations in AAAD paralogs leading to the convergent evolution of the derived aldehyde synthase activity. Applying knowledge learned from this study, we successfully engineered a shortened benzylisoquinoline alkaloid pathway to produce (S)-norcoclaurine in yeast. This work highlights the pliability of the AAAD fold that allows change of substrate selectivity and access to alternative catalytic mechanisms with only a few mutations.

Multifunctional lubricant additives derived from natural amino acids and methyl oleate

RSC Advances ◽  
2015 ◽  
Vol 5 (95) ◽  
pp. 77538-77544 ◽  
Author(s):  
Arukali Sammaiah ◽  
Korlipara V. Padmaja ◽  
Shiva Shanker Kaki ◽  
Rachapudi B. N. Prasad
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Methyl Oleate ◽  
Thioglycolic Acid ◽  
Methyl Esters ◽  
Lubricant Additives ◽  
Acid Methyl ◽  
Multifunctional Additives ◽  
Natural Amino Acids ◽  
Amino Acid Methyl Esters

Novel multifunctional additives were synthesized from methyl oleate via thioglycolic acid addition followed by condensation with different amino acid methyl esters.

scholarly journals High-Yield, Zero-Leakage Expression System with a Translational Switch Using Site-Specific Unnatural Amino Acid Incorporation

2013 ◽  
Vol 80 (5) ◽  
pp. 1718-1725 ◽  
Author(s):  
Masaomi Minaba ◽  
Yusuke Kato
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Unnatural Amino Acids ◽  
Mammalian Cells ◽  
Expression System ◽  
Trna Synthetase ◽  
Unnatural Amino Acid ◽  
Translational Efficiency ◽  
Site Specific ◽  
Content Type

ABSTRACTSynthetic biologists construct complex biological circuits by combinations of various genetic parts. Many genetic parts that are orthogonal to one another and are independent of existing cellular processes would be ideal for use in synthetic biology. However, our toolbox is still limited with respect to the bacteriumEscherichia coli, which is important for both research and industrial use. The site-specific incorporation of unnatural amino acids is a technique that incorporates unnatural amino acids into proteins using a modified exogenous aminoacyl-tRNA synthetase/tRNA pair that is orthogonal to any native pairs in a host and is independent from other cellular functions. Focusing on the orthogonality and independency that are suitable for the genetic parts, we designed novel AND gate and translational switches using the unnatural amino acid 3-iodo-l-tyrosine incorporation system inE. coli. A translational switch was turned on after addition of 3-iodo-l-tyrosine in the culture medium within minutes and allowed tuning of switchability and translational efficiency. As an application, we also constructed a gene expression system that produced large amounts of proteins under induction conditions and exhibited zero-leakage expression under repression conditions. Similar translational switches are expected to be applicable also for eukaryotes such as yeasts, nematodes, insects, mammalian cells, and plants.

Synthesis of Chiral N-(Purin-6-yl)amino Acid Derivatives by using Natural Amino Acids as Starting Materials

2012 ◽  
Vol 1 (3) ◽  
pp. 238-244 ◽  
Author(s):  
Hong-Ying Niu ◽  
Shi-Xia Bai ◽  
Shan Wu ◽  
Gui-Rong Qu ◽  
Hai-Ming Guo
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Derivatives ◽  
Natural Amino Acids

scholarly journals E. coli GyrA Tower Domain Interacts with QnrB1 Loop B and Plays an Important Role in QnrB1 Protection from Quinolone Inhibition

2021 ◽  
Author(s):  
Chunhui Chen ◽  
Yin Wang ◽  
Hidemasa Nakaminami ◽  
Eu Suk Kim ◽  
George A. Jacoby ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Amino Acids ◽  
Amino Acid ◽  
Dna Gyrase ◽  
Amino Acid Residues ◽  
Site Specific ◽  
E Coli ◽  
Small Deletion ◽  
Interaction Sites ◽  
Repeat Proteins

The Qnr pentapeptide repeat proteins interact with DNA gyrase and protect it from quinolone inhibition. The two external loops, particularly the larger loop B, of Qnr proteins are essential for quinolone protection of DNA gyrase. The specific QnrB1 interaction sites on DNA gyrase are not known. In this study, we investigated the interaction between GyrA and QnrB1 using site-specific photo crosslinking of QnrB1 loop B combined with mass spectrometry. We found that amino acid residues 286-298 on the Tower domain of GyrA interact with QnrB1 and play a key role in QnrB1 protection of gyrase from quinolone inhibition. Alanine replacement of arginine at residue 293 and a small deletion of amino acids 286-289 of GyrA resulted in a decrease in the QnrB1-mediated increase in quinolone MICs and also abolished the QnrB1 protection of purified DNA gyrase from ciprofloxacin inhibition.

scholarly journals Amino Acid Requirements of the Chinese Hamster Ovary Cell Metabolism during Recombinant Protein Production

2019 ◽  
Author(s):  
Bergthor Traustason
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Recombinant Protein ◽  
Recombinant Proteins ◽  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Scale Model ◽  
Amino Acid Requirements

SummaryMajority of biopharmaceutical drugs today are produced by Chinese hamster ovary (CHO) cells, which have been the standard industry host for the past decades. To produce and secrete a substantial amount of the target recombinant proteins the CHO cells must be provided with suitable growth conditions and provided with the necessary nutrients. Amino acids play a key role in this as the building blocks of proteins, playing important roles in a large number of metabolic pathways and being important sources of nitrogen as well as carbon under certain conditions. In this study exploratory analysis of the amino acid requirements of CHO cells was carried out using metabolic modelling approaches. Flux balance analysis was employed to evaluate the optimal distribution of fluxes in a genome-scale model of CHO cells to gain information on the cells’ metabolic response in silico.The results showed that providing non-essential amino acids (NEAAs) has a positive effect on CHO cell biomass production and that cysteine as well as tyrosine play a fundamental role in this. This implies that extracellular provision of NEAAs limits the extent of energy loss in amino acid biosynthetic pathways and renders additional reducing power available for other biological processes. Detailed analysis of the possible secretion and uptake of D-serine in the CHO model was also performed and its influence on the rest of the metabolism mapped out, which revealed results matching various existing literature. This is interesting since no mention of D-serine in regard to CHO cells was found in current literature, as well as the fact that this opens up the possibility of using the model for better understanding of certain disorders in higher up organisms that have been implicated with D-serine, such as motor neuron and cognitive degeneration. Finally, outcome from the model optimisation of different recombinant proteins demonstrated clearly how the difference in protein structure and size can influence the production outcome. These results show that systematic and model-based approaches have great potential for broad de novo exploration as well as being able to handle the cellular burden associated with the production of different types of recombinant protein.

scholarly journals Development of natural and unnatural amino acid delivery systems against hookworm infection

2020 ◽  
Vol 3 (1) ◽  
pp. 471-482 ◽  
Author(s):  
Stacey Bartlett ◽  
Mariusz Skwarczynski ◽  
Xin Xie ◽  
Istvan Toth ◽  
Alex Loukas ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Single Molecule ◽  
Cell Epitope ◽  
Unnatural Amino Acid ◽  
Nippostrongylus Brasiliensis ◽  
Peptide Fragments ◽  
Specific Pathogen ◽  
Hydrophobic Amino Acids ◽  
Natural Amino Acids

Peptide-based vaccines consist of short antigen fragments derived from a specific pathogen. Alone, these peptide fragments are poorly or non-immunogenic; however, when incorporated into a proper delivery system, they can trigger strong immune responses. To eliminate the need for toxic and often ineffective oral adjuvants, we designed single molecule-based self-adjuvating vaccines against hookworms using natural and unnatural hydrophobic amino acids. Two vaccine conjugates were synthesized, consisting of B-cell epitope p3, derived from the hookworm Na-APR-1 protein; universal T-helper peptide P25; and either double copies of unnatural lipoamino acid (2-amino-D,L-eicosanoic acid), or ten copies of the natural amino acid leucine. After challenge with the model hookworm, Nippostrongylus brasiliensis, mice orally immunized with the conjugates, but without adjuvant, generated antibody responses against the hookworm epitope, resulting in significantly reduced worm and egg burdens compared to control mice. We have demonstrated that vaccine nanoparticles composed exclusively of natural amino acids can be effective even when administered orally.

scholarly journals A basic motif in the N-terminal region of RAG1 enhances V(D)J recombination activity.

1997 ◽  
Vol 17 (8) ◽  
pp. 4544-4552 ◽  
Author(s):  
C J McMahan ◽  
M J Difilippantonio ◽  
N Rao ◽  
E Spanopoulou ◽  
D G Schatz
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Basic Amino Acid ◽  
Terminal Region ◽  
Recombination Reaction ◽  
Recombination Activity ◽  
Site Specific ◽  
Order Of Magnitude ◽  
Extrachromosomal Recombination ◽  
Enzymatic Machinery

The variable portions of antigen receptor genes are assembled from component gene segments by a site-specific recombination reaction known as V(D)J recombination. The RAG1 and RAG2 proteins are the critical lymphoid cell-specific components of the recombination enzymatic machinery and are responsible for site-specific DNA recognition and cleavage. Previous studies had defined a minimal, recombinationally active core region of murine RAG1 consisting of amino acids 384 to 1008 of the 1,040-residue RAG1 protein. No recombination function has heretofore been ascribed to any portion of the 383-amino-acid N-terminal region that is missing from the core, but it seems likely to be of functional significance, based on its evolutionary conservation. Using extrachromosomal recombination substrates, we demonstrate here that the N-terminal region enhances the recombination activity of RAG1 by up to an order of magnitude in a variety of cell lines. Deletion analysis localized a region of the N terminus critical for this effect to amino acids 216 to 238, and further mutagenesis demonstrated that a small basic amino acid motif (BIIa) in this region is essential for enhancing the activity of RAG1. Despite the fact that BIIa is important for the interaction of RAG1 with the nuclear localization factor Srp-1, it does not appear to enhance recombination by facilitating nuclear transport of RAG1. A variety of models for how this region stimulates the recombination activity of RAG1 are considered.

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