Examining the Structural and Chemical Flexibility of the Active Site Base, Lys-258, of Escherichia coli Aspartate Aminotransferase by Replacement with Unnatural Amino Acids

More than two decades ago a general method to genetically encode noncanonical or unnatural amino acids (NAAs) with diverse physical, chemical, or biological properties in bacteria, yeast, animals and mammalian cells was developed. More than 200 NAAs have been incorporated into recombinant proteins by means of non-endogenous aminoacyl-tRNA synthetase (aa-RS)/tRNA pair, an orthogonal pair, that directs site-specific incorporation of NAA encoded by a unique codon. The most established method to genetically encode NAAs in Escherichia coli is based on the usage of the desired mutant of Methanocaldococcus janaschii tyrosyl-tRNA synthetase (MjTyrRS) and cognate suppressor tRNA. The amber codon, the least-used stop codon in E. coli, assigns NAA. Until very recently the genetic code expansion technology suffered from a low yield of targeted proteins due to both incompatibilities of orthogonal pair with host cell translational machinery and the competition of suppressor tRNA with release factor (RF) for binding to nonsense codons. Here we describe the latest progress made to enhance nonsense suppression in E. coli with the emphasis on the improved expression vectors encoding for an orthogonal aa-RA/tRNA pair, enhancement of aa-RS and suppressor tRNA efficiency, the evolution of orthogonal EF-Tu and attempts to reduce the effect of RF1.

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An efficient system for incorporation of unnatural amino acids in response to the four-base codon AGGA in Escherichia coli

Biochimica et Biophysica Acta (BBA) - General Subjects ◽

10.1016/j.bbagen.2017.02.017 ◽

2017 ◽

Vol 1861 (11) ◽

pp. 3016-3023 ◽

Cited By ~ 8

Author(s):

Byeong Sung Lee ◽

Suyeon Kim ◽

Byoung Joon Ko ◽

Tae Hyeon Yoo

Keyword(s):

Escherichia Coli ◽

Amino Acids ◽

Unnatural Amino Acids ◽

Efficient System

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Crystal structures of the editing domain of Escherichia coli leucyl-tRNA synthetase and its complexes with Met and Ile reveal a lock-and-key mechanism for amino acid discrimination

Biochemical Journal ◽

10.1042/bj20051249 ◽

2006 ◽

Vol 394 (2) ◽

pp. 399-407 ◽

Cited By ~ 20

Author(s):

Yunqing Liu ◽

Jing Liao ◽

Bin Zhu ◽

En-Duo Wang ◽

Jianping Ding

Keyword(s):

Escherichia Coli ◽

Amino Acids ◽

Crystal Structures ◽

Active Site ◽

Binding Pocket ◽

Trna Synthetase ◽

Vital Role ◽

Common Mechanism ◽

Trna Synthetases ◽

Editing Domain

aaRSs (aminoacyl-tRNA synthetases) are responsible for the covalent linking of amino acids to their cognate tRNAs via the aminoacylation reaction and play a vital role in maintaining the fidelity of protein synthesis. LeuRS (leucyl-tRNA synthetase) can link not only the cognate leucine but also the nearly cognate residues Ile and Met to tRNALeu. The editing domain of LeuRS deacylates the mischarged Ile–tRNALeu and Met–tRNALeu. We report here the crystal structures of ecLeuRS-ED (the editing domain of Escherichia coli LeuRS) in both the apo form and in complexes with Met and Ile at 2.0 Å, 2.4 Å, and 3.2 Å resolution respectively. The editing active site consists of a number of conserved amino acids, which are involved in the precise recognition and binding of the noncognate amino acids. The substrate-binding pocket has a rigid structure which has an optimal stereochemical fit for Ile and Met, but has steric hindrance for leucine. Based on our structural results and previously available biochemical data, we propose that ecLeuRS-ED uses a lock-and-key mechanism to recognize and discriminate between the amino acids. Structural comparison also reveals that all subclass Ia aaRSs share a conserved structure core consisting of the editing domain and conserved residues at the editing active site, suggesting that these enzymes may use a common mechanism for the editing function.

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[Arg292 → val] or [Arg292 → leu] mutation enhances the reactivity of Escherichia, coli aspartate aminotransferase with aromatic amino acids

Biochemical and Biophysical Research Communications ◽

10.1016/0006-291x(89)92443-1 ◽

1989 ◽

Vol 159 (1) ◽

pp. 337-342 ◽

Cited By ~ 34

Author(s):

Hideyuki Hayashi ◽

Seiki Kuramitsu ◽

Yasushi Inoue ◽

Yoshimasa Morino ◽

Hiroyuki Kagamiyama

Keyword(s):

Escherichia Coli ◽

Amino Acids ◽

Aspartate Aminotransferase ◽

Aromatic Amino Acids

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2.8-.ANG.-resolution crystal structure of an active-site mutant of aspartate aminotransferase from Escherichia coli

Biochemistry ◽

10.1021/bi00446a030 ◽

1989 ◽

Vol 28 (20) ◽

pp. 8161-8167 ◽

Cited By ~ 74

Author(s):

Douglas L. Smith ◽

Steven C. Almo ◽

Michael D. Toney ◽

Dagmar Ringe

Keyword(s):

Crystal Structure ◽

Escherichia Coli ◽

Active Site ◽

Aspartate Aminotransferase

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Role of D-aminoacyl-tRNA deacylase beyond chiral proofreading as a cellular defense against glycine mischarging by AlaRS

eLife ◽

10.7554/elife.24001 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 19

Author(s):

Komal Ishwar Pawar ◽

Katta Suma ◽

Ayshwarya Seenivasan ◽

Santosh Kumar Kuncha ◽

Satya Brata Routh ◽

...

Keyword(s):

Escherichia Coli ◽

Amino Acids ◽

Active Site ◽

Unknown Function ◽

Physiological Importance ◽

Cellular Defense

Strict L-chiral rejection through Gly-cisPro motif during chiral proofreading underlies the inability of D-aminoacyl-tRNA deacylase (DTD) to discriminate between D-amino acids and achiral glycine. The consequent Gly-tRNAGly ‘misediting paradox’ is resolved by EF-Tu in the cell. Here, we show that DTD’s active site architecture can efficiently edit mischarged Gly-tRNAAla species four orders of magnitude more efficiently than even AlaRS, the only ubiquitous cellular checkpoint known for clearing the error. Also, DTD knockout in AlaRS editing-defective background causes pronounced toxicity in Escherichia coli even at low-glycine levels which is alleviated by alanine supplementation. We further demonstrate that DTD positively selects the universally invariant tRNAAla-specific G3•U70. Moreover, DTD’s activity on non-cognate Gly-tRNAAla is conserved across all bacteria and eukaryotes, suggesting DTD’s key cellular role as a glycine deacylator. Our study thus reveals a hitherto unknown function of DTD in cracking the universal mechanistic dilemma encountered by AlaRS, and its physiological importance.

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