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

eLife ◽  
2017 ◽  
Vol 6 ◽  
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.

scholarly journals Roles of the C-Terminal Amino Acids of Non-Hexameric Helicases: Insights from Escherichia coli UvrD

2021 ◽  
Vol 22 (3) ◽  
pp. 1018
Author(s):  
Hiroaki Yokota
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Amino Acid ◽  
Single Molecule ◽  
Underlying Mechanism ◽  
Amino Acid Sequences ◽  
E Coli ◽  
X Ray Crystallography ◽  
Terminal Amino

Helicases are nucleic acid-unwinding enzymes that are involved in the maintenance of genome integrity. Several parts of the amino acid sequences of helicases are very similar, and these quite well-conserved amino acid sequences are termed “helicase motifs”. Previous studies by X-ray crystallography and single-molecule measurements have suggested a common underlying mechanism for their function. These studies indicate the role of the helicase motifs in unwinding nucleic acids. In contrast, the sequence and length of the C-terminal amino acids of helicases are highly variable. In this paper, I review past and recent studies that proposed helicase mechanisms and studies that investigated the roles of the C-terminal amino acids on helicase and dimerization activities, primarily on the non-hexermeric Escherichia coli (E. coli) UvrD helicase. Then, I center on my recent study of single-molecule direct visualization of a UvrD mutant lacking the C-terminal 40 amino acids (UvrDΔ40C) used in studies proposing the monomer helicase model. The study demonstrated that multiple UvrDΔ40C molecules jointly participated in DNA unwinding, presumably by forming an oligomer. Thus, the single-molecule observation addressed how the C-terminal amino acids affect the number of helicases bound to DNA, oligomerization, and unwinding activity, which can be applied to other helicases.

Peroxovanadates and Its Bio-Mimicking Relation with Vanadium Haloperoxidases

2016 ◽  
pp. 197-219
Author(s):  
Pranjal Saikia ◽  
Saitanya Kumar Bharadwaj ◽  
Abu Taleb Miah
Keyword(s):  
Amino Acids ◽  
Active Site ◽  
High Efficiency ◽  
Recombinant Enzymes ◽  
Vanadium Compounds ◽  
Reaction Conditions ◽  
The Cost ◽  
Major Emphasis ◽  
Protein Active Site

Vanadium Haloperoxidases (VHPOs) have been used in a variety of biotransformations showing remarkable stereoselectivity and regiospecificity. The high efficiency of the enzyme is influenced by the protein active site and the role of certain amino acids in activation of vanadium(V)-bound peroxide for halide oxidation. The use of natural or recombinant enzymes, or biomimetic vanadium compounds brings up issues regarding the cost of production and reaction conditions. In this chapter, the primary intent is to provide a simple and clear picture of functional mimicking nature of peroxovanadium compounds with haloperoxidases enzymes to the readers. Major emphasis would be given to examine the reactivity of the vanadium haloperoxidases with mechanism.

Mannan-Degrading Enzymes fromCellulomonas fimi

1999 ◽  
Vol 65 (6) ◽  
pp. 2598-2605 ◽  
Author(s):  
Dominik Stoll ◽  
Henrik Stålbrand ◽  
R. Antony J. Warren
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Unknown Function ◽  
Glycosyl Hydrolases ◽  
Intracellular Enzyme ◽  
Cellulomonas Fimi ◽  
Catalytic Module ◽  
Mannanase Activity ◽  
Homology Module ◽  
Active Fragments

ABSTRACT The genes man26a and man2A fromCellulomonas fimi encode mannanase 26A (Man26A) and β-mannosidase 2A (Man2A), respectively. Mature Man26A is a secreted, modular protein of 951 amino acids, comprising a catalytic module in family 26 of glycosyl hydrolases, an S-layer homology module, and two modules of unknown function. Exposure of Man26A produced byEscherichia coli to C. fimi protease generates active fragments of the enzyme that correspond to polypeptides with mannanase activity produced by C. fimi during growth on mannans, indicating that it may be the only mannanase produced by the organism. A significant fraction of the Man26A produced by C. fimi remains cell associated. Man2A is an intracellular enzyme comprising a catalytic module in a subfamily of family 2 of the glycosyl hydrolases that at present contains only mammalian β-mannosidases.

scholarly journals The mechanism of action of dd-peptidases: the role of Threonine-299 and -301 in the Streptomyces R61 dd-peptidase

1994 ◽  
Vol 301 (2) ◽  
pp. 477-483 ◽  
Author(s):  
J M Wilkin ◽  
A Dubus ◽  
B Joris ◽  
J M Frère
Keyword(s):  
Amino Acids ◽  
Active Site ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Peptide Substrate ◽  
Binding Properties ◽  
Beta Lactamases ◽  
Penicillin Binding Proteins ◽  
Active Site Cavity

The side chains of residues Thr299 and Thr301 in the Streptomyces R61 DD-peptidase have been modified by site-directed mutagenesis. These amino acids are part of a beta-strand which forms a wall of the active-site cavity. Thr299 corresponds to the second residue of the Lys-Thr(Ser)-Gly triad, highly conserved in active-site beta-lactamases and penicillin-binding proteins (PBPs). Modification of Thr301 resulted only in minor alterations of the catalytic and penicillin-binding properties of the enzyme. No selective decrease of the rate of acylation was observed for any particular class of compounds. By contrast, the loss of the hydroxy group of the residue in position 299 yielded a seriously impaired enzyme. The rates of inactivation by penicillins were decreased 30-50-fold, whereas the reactions with cephalosporins were even more affected. The efficiency of hydrolysis against the peptide substrate was also seriously decreased. More surprisingly, the mutant was completely unable to catalyse transpeptidation reactions. The conservation of an hydroxylated residue in this position in PBPs is thus easily explained by these results.

scholarly journals Purification of the STBenterotoxin of Escherichia coli and the role of selected amino acids on its secretion, stability and toxicity

1993 ◽  
Vol 7 (6) ◽  
pp. 1026-1026
Author(s):  
Lawrence A. Dreyfus ◽  
Robert G. Urban ◽  
Shannon C. Whipp ◽  
Clive Slaughter ◽  
Kathy Tachias ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acids

scholarly journals Role of the sRNA GcvB in regulation of cycA in Escherichia coli

Microbiology ◽  
2009 ◽  
Vol 155 (1) ◽  
pp. 106-114 ◽  
Author(s):  
Sarah C. Pulvermacher ◽  
Lorraine T. Stauffer ◽  
George V. Stauffer
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Minimal Medium ◽  
Luria Bertani ◽  
Cell Physiology ◽  
Rt Pcr ◽  
Luria Bertani Broth ◽  
Increased Sensitivity ◽  
Transcriptional Fusions

In Escherichia coli, the gcvB gene encodes a small non-translated RNA that regulates several genes involved in transport of amino acids and peptides (including sstT, oppA and dppA). Microarray analysis identified cycA as an additional regulatory target of GcvB. The cycA gene encodes a permease for the transport of glycine, d-alanine, d-serine and d-cycloserine. RT-PCR confirmed that GcvB and the Hfq protein negatively regulate cycA mRNA in cells grown in Luria–Bertani broth. In addition, deletion of the gcvB gene resulted in increased sensitivity to d-cycloserine, consistent with increased expression of cycA. A cycA : : lacZ translational fusion confirmed that GcvB negatively regulates cycA expression in Luria–Bertani broth and that Hfq is required for the GcvB effect. GcvB had no effect on cycA : : lacZ expression in glucose minimal medium supplemented with glycine. However, Hfq still negatively regulated the fusion in the absence of GcvB. A set of transcriptional fusions of cycA to lacZ identified a sequence in cycA necessary for regulation by GcvB. Analysis of GcvB identified a region complementary to this region of cycA mRNA. However, mutations predicted to disrupt base-pairing between cycA mRNA and GcvB did not alter expression of cycA : : lacZ. A model for GcvB function in cell physiology is discussed.

Role of tryptophan 248 in the active site of tryptophanase from Escherichia coli

1990 ◽  
Vol 173 (2) ◽  
pp. 756-762 ◽  
Author(s):  
Yasushi Kawata ◽  
Nobuharu Tsujimoto ◽  
Shunsuke Tani ◽  
Tomohiro Mizobata ◽  
Masanobu Tokushige
Keyword(s):  
Escherichia Coli ◽  
Active Site

scholarly journals 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

2006 ◽  
Vol 394 (2) ◽  
pp. 399-407 ◽  
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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