Article
Chemistry, Multidisciplinary
Adam T. Beattie, Daniel L. Dunkelmann, Jason W. Chin
Summary: Mutually orthogonal aminoacyl transfer RNA synthetase/transfer RNA pairs are essential for encoding non-canonical amino acids into proteins. In this study, we discovered quintuply orthogonal pyrrolysyl-tRNA synthetase (PylRS)/pyrrolysyl-tRNA (tRNA(Pyl)) pairs and identified empirical sequence identity thresholds for mutual orthogonality. By testing various pairs and engineering specificities, we were able to create 924 mutually orthogonal PylRS/tRNA(Pyl) pairs, 1324 triply orthogonal pairs, 128 quadruply orthogonal pairs, and 8 quintuply orthogonal pairs.
Article
Biochemistry & Molecular Biology
Natalie Krahn, Jingji Zhang, Sergey Melnikov, Jeffery M. Tharp, Alessandra Villa, Armaan Patel, Rebecca J. Howard, Haben Gabir, Trushar R. Patel, Jorg Stetefeld, Joseph Puglisi, Dieter Soll
Summary: Protein translation is achieved through tRNA aminoacylation and ribosomal elongation. This study explores the tRNA identity elements for a Delta pylSn tRNA(Pyl) and identifies five key elements necessary for MaPylRS activity.
NUCLEIC ACIDS RESEARCH
(2023)
Article
Biochemical Research Methods
Qunfeng Zhang, Wenlong Zheng, Zhongdi Song, Qiang Zhang, Lirong Yang, Jianping Wu, Jianping Lin, Gang Xu, Haoran Yu
Summary: This study developed machine learning models to predict the substrate specificity of PylRS for novel NCAAs. The models showed high accuracy and provided a framework for expanding the substrate scope of PylRS variants and developing machine learning models for other PylRS variants.
ACS SYNTHETIC BIOLOGY
(2023)
Article
Biotechnology & Applied Microbiology
Nikolaj G. Koch, Tobias Baumann, Nediljko Budisa
Summary: Introducing non-canonical amino acids (ncAAs) through engineered orthogonal pairs of aminoacyl-tRNA synthetases and tRNAs is a useful tool for expanding the genetic code, but often limited by low yields of chemically modified target proteins. The solubility and folding of engineered enzymes can be a bottleneck for the production of ncAA-containing proteins in vivo, with strategies such as solubility tags improving enzyme solubility and translation efficiency. These methods enhance protein production with engineered PylRS variants and even wild-type enzymes, showing significant efficiency improvements.
FRONTIERS IN BIOENGINEERING AND BIOTECHNOLOGY
(2021)
Article
Biochemical Research Methods
Jessica T. Stieglitz, Priyanka Lahiri, Matthew Stout, James A. Van Deventer
Summary: Archaeal pyrrolysyl-tRNA synthetases (PylRSs) have been used to genetically encode over 200 distinct noncanonical amino acids (ncAAs) in proteins in Escherichia coli and mammalian cells. This study demonstrates the potential of using Methanomethylophilus alvus PylRS (MaPylRS) in yeast to incorporate ncAAs into proteins. The addition of MaPylRS to the toolkit of translation machinery in Saccharomyces cerevisiae opens up possibilities for expanding the range of genetically encodable ncAAs.
ACS SYNTHETIC BIOLOGY
(2022)
Article
Chemistry, Multidisciplinary
Yi-Hui Wang, Mu-Lung Jian, Pei-Jung Chen, Jo-Chu Tsou, Le P. Truong, Yane-Shih Wang
Summary: This study explores the application of expanding genetic codes in developing protein cage-based delivery systems, utilizing evolved Methanosarcina mazei pyrrolysyl-tRNA synthetase (PylRS) and tRNA(Pyl) to recognize para-substituted phenylalanine analogs. The engineered variants successfully incorporate p-azido-l-phenylalanine (AzF) into human heavy chain ferritin (Ftn), demonstrating the potential for site-specific drug loading in protein nanocages.
FRONTIERS IN CHEMISTRY
(2021)
Article
Biochemistry & Molecular Biology
David G. Schwark, Margaret A. Schmitt, John D. Fisk
Summary: Genetic code expansion has focused on reassigning amber stop codons to insert non-canonical amino acids into proteins. Efforts have been made to use evolved aminoacyl tRNA synthetase variants to incorporate multiple non-canonical amino acids in response to sense codons. A new highly efficient variant of an orthogonal tRNA/aaRS pair was evolved to activate and incorporate tyrosine, rivaling the efficiency of the wild-type pair.
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
(2021)
Article
Biochemistry & Molecular Biology
Erol C. Vatansever, Kai S. Yang, Zhi Zachary Geng, Yuchen Qiao, Pingwei Li, Shiqing Xu, Wenshe Ray Liu
Summary: Researchers have designed a PylRS mutant, oClFRS, which efficiently catalyzes the genetic incorporation of o-substituted phenylalanines and have elucidated its structure and function. The binding of o-ClF in the active site of oClFRS involves two halogen bonds, which are crucial for its activity.
JOURNAL OF MOLECULAR BIOLOGY
(2022)
Article
Biochemistry & Molecular Biology
Li -Tao Guo, Kazuaki Amikura, Han -Kai Jiang, Takahito Mukai, Xian Fu, Yane-Shih Wang, Patrick O'Donoghue, Dieter Soell, Jeffery M. Tharp
Summary: The pyrrolysyl-tRNA synthetase (PylRS) is a major route to install noncanonical amino acids into proteins in living cells due to its tolerance for diverse amino acid substrates and orthogonality in multiple organisms. A novel class of PylRS enzymes was identified in a subset of methanogenic archaea, lacking the N-terminal tRNA-binding domain yet remaining active and orthogonal in bacteria and eukaryotes. Molecular phylogeny analysis revealed the coevolutionary history of PylRS and tRNAPyl, with the emergence of tRNAPyl sequences containing unique discriminator bases that may enable further genetic code expansion efforts.
JOURNAL OF BIOLOGICAL CHEMISTRY
(2022)
Article
Biochemistry & Molecular Biology
Chia-Chuan Cho, Lauren R. Blankenship, Xinyu Ma, Shiqing Xu, Wenshe Liu
Summary: The study investigated the amber suppression-based noncanonical amino acid mutagenesis technique in both basic and applied research, discovering a new cleavage mechanism of MmPylRS and stabilizing MmPylRS by introducing the P188G mutation, enabling enhanced incorporation of BocK and other noncanonical amino acids.
JOURNAL OF MOLECULAR BIOLOGY
(2022)
Article
Biochemistry & Molecular Biology
Jonathan T. Fischer, Dieter Soll, Jeffery M. Tharp
Summary: This study evolved MaPylRS enzyme using PANCE technology to obtain a highly active variant, PylRS(opt), which exhibits high activity and selectivity towards multiple amino acid derivatives and can be used to enhance the activity of other PylRS constructs.
FRONTIERS IN MOLECULAR BIOSCIENCES
(2022)
Article
Biochemistry & Molecular Biology
Jin-Tao Wang, Jing-Bo Zhou, Xue-Ling Mao, Li Zhou, Meirong Chen, Wenhua Zhang, En-Duo Wang, Xiao-Long Zhou
Summary: This study reveals the crucial role of t(6)A modification in tRNA(Ile) aminoacylation and codon decoding, as well as the commonality and diversity in substrate recognition by eukaryotic KEOPS.
NUCLEIC ACIDS RESEARCH
(2022)
Article
Multidisciplinary Sciences
Kommireddy Vasu, Iyappan Ramachandiran, Fulvia Terenzi, Debjit Khan, Arnab China, Krishnendu Khan, Aayushi Chechi, Camelia Baleanu-Gogonea, Valentin Gogonea, Paul L. Fox
Summary: The study reveals the critical role of the ZBD in determining the activity of ProRS and the stability of GluProRS, with disruptions of the ZBD leading to a reduction in GluProRS canonical function.
Article
Genetics & Heredity
Takahito Mukai, Kazuaki Amikura, Xian Fu, Dieter Soell, Ana Crnkovic
Summary: This study provides a comprehensive bioinformatic analysis of the diversity of indirect cysteine encoding systems. The results shed new light on the variations in SepRS and SepCysS enzymes, adaptation to lifestyle and habitat, and provide new information on the evolution of the genetic code.
FRONTIERS IN GENETICS
(2022)
Article
Biochemistry & Molecular Biology
Meriem Elkolli, Hayet Elkolli, Manawwer Alam, Yacine Benguerba
Summary: The misuse and overuse of antibiotics have led to antibiotic resistance. However, terpene-rich essential oils provide a potential alternative for antimicrobial agents. In this study, we demonstrated the antibacterial activity of three plant essential oils and identified their binding energies with a critical enzyme involved in bacterial protein synthesis through molecular docking.
JOURNAL OF BIOMOLECULAR STRUCTURE & DYNAMICS
(2023)