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 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.

scholarly journals Amino acid substitutions and alteration in cation specificity in the melibiose carrier of Escherichia coli.

1988 ◽  
Vol 263 (28) ◽  
pp. 14276-14280 ◽  
Author(s):  
T Kawakami ◽  
Y Akizawa ◽  
T Ishikawa ◽  
T Shimamoto ◽  
M Tsuda ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Substitutions

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.

Structural and functional consequences of single amino acid substitutions in the pyrimidine base binding pocket of Escherichia coli CMP kinase

FEBS Journal ◽  
2007 ◽  
Vol 274 (13) ◽  
pp. 3363-3373 ◽  
Author(s):  
Augustin Ofiteru ◽  
Nadia Bucurenci ◽  
Emil Alexov ◽  
Thomas Bertrand ◽  
Pierre Briozzo ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Binding Pocket ◽  
Single Amino Acid ◽  
Amino Acid Substitutions ◽  
Pyrimidine Base ◽  
Functional Consequences

scholarly journals Investigation of the Role of Electrostatic Charge in Activation of the Escherichia coli Response Regulator CheY

2003 ◽  
Vol 185 (21) ◽  
pp. 6385-6391 ◽  
Author(s):  
Jenny G. Smith ◽  
Jamie A. Latiolais ◽  
Gerald P. Guanga ◽  
Sindhura Citineni ◽  
Ruth E. Silversmith ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Signal Transduction ◽  
Amino Acid ◽  
Active Site ◽  
Response Regulator ◽  
Residual Activity ◽  
Phosphoryl Group ◽  
Sensor Kinase ◽  
Amino Acid Substitutions ◽  
Flagellar Rotation

ABSTRACT In a two-component regulatory system, an important means of signal transduction in microorganisms, a sensor kinase phosphorylates a response regulator protein on an aspartyl residue, resulting in activation. The active site of the response regulator is highly charged (containing a lysine, the phosphorylatable aspartate, two additional aspartates involved in metal binding, and an Mg2+ ion), and introduction of the dianionic phosphoryl group results in the repositioning of charged moieties. Furthermore, substitution of one of the Mg2+-coordinating aspartates with lysine or arginine in the Escherichia coli chemotaxis response regulator CheY results in phosphorylation-independent activation. In order to examine the consequences of altered charge distribution for response regulator activity and to identify possible additional amino acid substitutions that result in phosphorylation-independent activation, we made 61 CheY mutants in which residues close to the site of phosphorylation (Asp57) were replaced by various charged amino acids. Most substitutions (47 of 61) resulted in the complete loss of CheY activity, as measured by the inability to support clockwise flagellar rotation. However, 10 substitutions, all introducing a new positive charge, resulted in the loss of chemotaxis but in the retention of some clockwise flagellar rotation. Of the mutants in this set, only the previously identified CheY13DK and CheY13DR mutants displayed clockwise activity in the absence of the CheA sensor kinase. The absence of negatively charged substitution mutants with residual activity suggests that the introduction of additional negative charges into the active site is particularly deleterious for CheY function. Finally, the spatial distribution of positions at which amino acid substitutions are functionally tolerated or not tolerated is consistent with the presently accepted mechanism of response regulator activation and further suggests a possible role for Met17 in signal transduction by CheY.

scholarly journals Mutations in the uncE gene affecting assembly of the c-subunit of the adenosine triphosphatase of Escherichia coli

1983 ◽  
Vol 211 (3) ◽  
pp. 717-726 ◽  
Author(s):  
D A Jans ◽  
A L Fimmel ◽  
L Langman ◽  
L B James ◽  
J A Downie ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Cell Membrane ◽  
Dna Sequences ◽  
Adenosine Triphosphatase ◽  
Gene Dosage ◽  
Base Change ◽  
Amino Acid Substitutions ◽  
Helical Hairpin ◽  
C Subunit

The amino acid substitutions in the mutant c-subunits of Escherichia coli F1F0-ATPase coded for by the uncE429, uncE408 and uncE463 alleles affect the incorporation of these proteins into the cell membrane. The DNA sequence of the uncE429 allele differed from normal in that a G leads to A base change occurred at nucleotide 68 of the uncE gene, resulting in glycine being replaced by aspartic acid at position 23 in the c-subunit. The uncE408 and uncE463 mutant DNA sequences were identical and differed from normal in that a C leads to T base change occurred at nucleotide 91 of the uncE gene, resulting in leucine being replaced by phenylalanine at position 31 in the c-subunit. An increased gene dosage of the uncE408 or uncE463 alleles resulted in the incorporation into the membranes of the mutant c-subunits. The results are discussed in terms of the ‘Helical Hairpin Hypothesis’ of Engelman & Steitz [(1981) Cell 23,411-422].

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