Role of the .beta.-phosphate and .gamma.-phosphate interchange reaction of adenosine triphosphate in amino acid discrimination by valyl- and methionyl-tRNA synthetases from Escherichia coli

Biochemistry ◽  
1981 ◽  
Vol 20 (2) ◽  
pp. 385-391 ◽  
Author(s):  
Linda Tombras Smith ◽  
Mildred Cohn
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Adenosine Triphosphate ◽  
Trna Synthetases ◽  
Interchange Reaction

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.

scholarly journals Effects of Amino Acid Substitutions at Conserved and Acidic Residues within Region 1.1 of Escherichia coli ς70

2000 ◽  
Vol 182 (1) ◽  
pp. 221-224 ◽  
Author(s):  
Christina Wilson Bowers ◽  
Andrea McCracken ◽  
Alicia J. Dombroski
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Aspartic Acid ◽  
Transcription Initiation ◽  
Amino Acid Substitutions ◽  
Conserved Residues ◽  
Acidic Residues

ABSTRACT Amino acid substitutions in Escherichia coliς70 were generated and characterized in an analysis of the role of region 1.1 in transcription initiation. Several acidic and conserved residues are tolerant of substitution. However, replacement of aspartic acid 61 with alanine results in inactivity caused by structural and functional thermolability.

scholarly journals The role of synonymous mutations in the evolution of TEM β-lactamase genes

2021 ◽  
Author(s):  
Mohammad Faheem ◽  
Charles J. Zhang ◽  
Monica N. Morris ◽  
Juergen Pleiss ◽  
Peter Oelschlaeger
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Disc Diffusion ◽  
Nucleotide Change ◽  
Nonsynonymous Mutation ◽  
Synonymous Mutations ◽  
Extended Spectrum ◽  
Genes Encoding ◽  
Variant Genes

Nonsynonymous mutations are well documented in TEM β-lactamases. The resulting amino acid changes often alter the conferred phenotype from broad spectrum (2b) conferred by TEM-1 to extended spectrum (2be), inhibitor resistant (2br), or both extended spectrum and inhibitor resistant (2ber). The encoding blaTEM genes also deviate in numerous synonymous mutations, which are not well understood. blaTEM-3 (2be), blaTEM-33 (2br), and blaTEM-109 (2ber) were studied in comparison to blaTEM-1. blaTEM-33 was chosen for more detailed studies, because it deviates from blaTEM-1 by a single nonsynonymous mutation and three additional, synonymous mutations. Genes encoding the enzymes with only nonsynonymous or all, including synonymous, mutations plus all permutations between blaTEM-1 and blaTEM-33 were expressed in Escherichia coli cells. In disc diffusion assays, genes encoding TEM-3, TEM-33, and TEM-109 with all synonymous mutations resulted in higher resistance levels than genes without synonymous mutations. Disc diffusion assays with the 16 genes carrying all possible nucleotide change combinations between blaTEM-1 and blaTEM-33 indicated different susceptibilities for different variants. Nucleotide BLAST searches did not identify genes without synonymous mutations but some without nonsynonymous mutations. Energies of possible secondary mRNA structures calculated with mfold are generally higher with synonymous mutations, suggesting that their role could be to destabilize the mRNA and facilitate its unfolding for efficient translation. In summary, our data indicate that transitions from blaTEM-1 to other variant genes by simply acquiring the nonsynonymous mutations is not favored. Instead, synonymous mutations seem to support the transition to other variant genes with nonsynonymous mutations leading to different phenotypes.

scholarly journals Functional role of catalytic triad and oxyanion hole-forming residues on enzyme activity of Escherichia coli thioesterase I/protease I/phospholipase L1

2006 ◽  
Vol 397 (1) ◽  
pp. 69-76 ◽  
Author(s):  
Li-Chiun Lee ◽  
Ya-Lin Lee ◽  
Ruey-Jyh Leu ◽  
Jei-Fu Shaw
Keyword(s):  
Escherichia Coli ◽  
Enzyme Activity ◽  
Amino Acid ◽  
Water Molecule ◽  
Acyl Chain ◽  
Residual Activity ◽  
Catalytic Triad ◽  
The Other ◽  
Oxyanion Hole

Escherichia coli TAP (thioesterase I, EC 3.1.2.2) is a multifunctional enzyme with thioesterase, esterase, arylesterase, protease and lysophospholipase activities. Previous crystal structural analyses identified its essential amino acid residues as those that form a catalytic triad (Ser10-Asp154-His157) and those involved in forming an oxyanion hole (Ser10-Gly44-Asn73). To gain an insight into the biochemical roles of each residue, site-directed mutagenesis was employed to mutate these residues to alanine, and enzyme kinetic studies were conducted using esterase, thioesterase and amino-acid-derived substrates. Of the residues, His157 is the most important, as it plays a vital role in the catalytic triad, and may also play a role in stabilizing oxyanion conformation. Ser10 also plays a very important role, although the small residual activity of the S10A variant suggests that a water molecule may act as a poor substitute. The water molecule could possibly be endowed with the nucleophilic-attacking character by His157 hydrogen-bonding. Asp154 is not as essential compared with the other two residues in the triad. It is close to the entrance of the substrate tunnel, therefore it predominantly affects substrate accessibility. Gly44 plays a role in stabilizing the oxyanion intermediate and additionally in acyl-enzyme-intermediate transformation. N73A had the highest residual enzyme activity among all the mutants, which indicates that Asn73 is not as essential as the other mutated residues. The role of Asn73 is proposed to be involved in a loop75–80 switch-move motion, which is essential for the accommodation of substrates with longer acyl-chain lengths.

scholarly journals Phospho-N-Acetyl-Muramyl-Pentapeptide Translocase from Escherichia coli: Catalytic Role of Conserved Aspartic Acid Residues

2004 ◽  
Vol 186 (6) ◽  
pp. 1747-1757 ◽  
Author(s):  
Adrian J. Lloyd ◽  
Philip E. Brandish ◽  
Andrea M. Gilbey ◽  
Timothy D. H. Bugg
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Aspartic Acid ◽  
Catalytic Mechanism ◽  
Partial Purification ◽  
Sequence Alignments ◽  
Structural Environment ◽  
Essential Enzyme ◽  
Covalent Intermediate

ABSTRACT Phospho-N-acetyl-muramyl-pentapeptide translocase (translocase 1) catalyzes the first of a sequence of lipid-linked steps that ultimately assemble the peptidoglycan layer of the bacterial cell wall. This essential enzyme is the target of several natural product antibiotics and has recently been the focus of antimicrobial drug discovery programs. The catalytic mechanism of translocase 1 is believed to proceed via a covalent intermediate formed between phospho-N-acetyl-muramyl-pentapeptide and a nucleophilic amino acid residue. Amino acid sequence alignments of the translocase 1 family and members of the related transmembrane phosphosugar transferase superfamily revealed only three conserved residues that possess nucleophilic side chains: the aspartic acid residues D115, D116, and D267. Here we report the expression and partial purification of Escherichia coli translocase 1 as a C-terminal hexahistidine (C-His6) fusion protein. Three enzymes with the site-directed mutations D115N, D116N, and D267N were constructed, expressed, and purified as C-His6 fusions. Enzymatic analysis established that all three mutations eliminated translocase 1 activity, and this finding verified the essential role of these residues. By analogy with the structural environment of the double aspartate motif found in prenyl transferases, we propose a model whereby D115 and D116 chelate a magnesium ion that coordinates with the pyrophosphate bridge of the UDP-N-acetyl-muramyl-pentapeptide substrate and in which D267 therefore fulfills the role of the translocase 1 active-site nucleophile.

Inhibition of Escherichia coli RNAp by T7 Gp2 Protein: Role of Negatively Charged Strip of Amino Acid Residues in Gp2

2011 ◽  
Vol 407 (5) ◽  
pp. 623-632 ◽  
Author(s):  
Carol Sheppard ◽  
Beatriz Cámara ◽  
Andrey Shadrin ◽  
Natalia Akulenko ◽  
Minhao Liu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Residues

scholarly journals Membrane topology of the Escherichia coli γ-aminobutyrate transporter: implications on the topography and mechanism of prokaryotic and eukaryotic transporters from the APC superfamily

1998 ◽  
Vol 336 (1) ◽  
pp. 69-76 ◽  
Author(s):  
Liaoyuan A. HU ◽  
Steven C. KING
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Membrane Topology ◽  
Amino Acid Transporters ◽  
Cationic Amino Acid ◽  
Transmembrane Segments ◽  
Bacteria And Fungi ◽  
Significant Region ◽  
Hydropathy Analysis

The Escherichia coli γ-aminobutyric acid permease (GabP) is a plasma membrane protein from the amine–polyamine–choline (APC) superfamily. On the basis of hydropathy analysis, transporters from this family are thought to contain 12, 13 or 14 transmembrane domains. We have experimentally analysed the topography of GabP by using the cytoplasmically active LacZ (β-galactosidase) and the periplasmically active PhoA (alkaline phosphatase) as complementary topological sensors. The enzymic activities of 32 GabP–LacZ hybrids and 43 GabP–PhoA hybrids provide mutually reinforcing lines of evidence that the E. coliGabP contains 12 transmembrane segments that traverse the membrane in a zig-zag fashion with both N- and C-termini facing the cytoplasm. Interestingly, the resulting model predicts that the functionally important ‘consensus amphipathic region ’ (CAR) [Hu and King (1998) Biochem. J. 330, 771–776] is at least partly membrane-embedded in many amino acid transporters from bacteria and fungi, in contrast with the apparent situation in mouse cationic amino acid transporters (MCATs), in which this kinetically significant region is thought to be fully cytoplasmic [Sophianopoulou and Diallinas (1995) FEMS Microbiol. Rev. 16, 53–75]. To the extent that conserved domains serve similar functions, the resolution of this topological disparity stands to have family-wide implications on the mechanistic role of the CAR. The consensus transmembrane structure derived from this analysis of GabP provides a foundation for predicting the topological disposition of the CAR and other functionally important domains that are conserved throughout the APC transporter superfamily.

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