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 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 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.

Expression and partial purification of human pro-opiomelanocortin in Escherichia coli

1989 ◽  
Vol 3 (2) ◽  
pp. 105-112 ◽  
Author(s):  
T. S. Grewal ◽  
P. J. Lowry ◽  
D. Savva
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Fusion Protein ◽  
Western Blot ◽  
Blot Analysis ◽  
Expression Vectors ◽  
Partial Purification ◽  
Amino Acid Residues ◽  
E Coli ◽  
Dna Fragment

ABSTRACT A large portion of the human pro-opiomelanocortin (POMC) peptide corresponding to amino acid residues 59–241 has been cloned and expressed in Escherichia coli. A 1·0 kb DNA fragment encoding this peptide was cloned into the expression vectors pUC8 and pUR291. Plasmid pJMBG51 (a pUC8 recombinant) was found to direct the expression of a 24 kDa peptide. The recombinant pUR291 (pJMBG52) was shown to produce a β-galactosidase fusion protein of 140 kDa. Western blot analysis showed that both the 24 kDa and 140 kDa peptides are recognized by antibodies raised against POMC-derived peptides. The β-galactosidase fusion protein has been partially purified from crude E. coli cell lysates using affinity chromatography on p-aminobenzyl-1-thio-β-d-galactopyranoside agarose.

Developing structure-based models to predict substrate specificity of D-group (Type II) molybdenum enzymes: application to a molybdo-enzyme of unknown function from Archaeoglobus fulgidus

2006 ◽  
Vol 34 (1) ◽  
pp. 118-121 ◽  
Author(s):  
E.J. Dridge ◽  
D.J. Richardson ◽  
R.J. Lewis ◽  
C.S. Butler
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Sequence Analysis ◽  
Archaeoglobus Fulgidus ◽  
Substrate Selectivity ◽  
Type Ii ◽  
Amino Acid Residues ◽  
Sequence Alignments ◽  
X Ray ◽  
Group Type

The AF0174–AF0176 gene cluster in Archaeoglobus fulgidus encodes a putative oxyanion reductase of the D-type (Type II) family of molybdo-enzymes. Sequence analysis reveals that the catalytic subunit AF0176 shares low identity (31–32%) and similarity (41–42%) to both NarG and SerA, the catalytic components of the respiratory nitrate and selenate reductases respectively. Consequently, predicting the oxyanion substrate selectivity of AF0176 has proved difficult based solely on sequence alignments. In the present study, we have modelled both AF0176 and SerA on the recently determined X-ray structure of the NAR (nitrate reductase) from Escherichia coli and have identified a number of key amino acid residues, conserved in all known NAR sequences, including AF0176, that we speculate may enhance selectivity towards trigonal planar (NO3−) rather than tetrahedral (SeO42− and ClO4−) substrates.

scholarly journals Site-directed mutagenesis of class I HLA genes. Role of glycosylation in surface expression and functional recognition.

1987 ◽  
Vol 166 (5) ◽  
pp. 1329-1350 ◽  
Author(s):  
J A Barbosa ◽  
J Santos-Aguado ◽  
S J Mentzer ◽  
J L Strominger ◽  
S J Burakoff ◽  
...  
Keyword(s):  
Amino Acid ◽  
Gene Transfer ◽  
Aspartic Acid ◽  
Human Cell ◽  
Surface Expression ◽  
Carbohydrate Moiety ◽  
Class I ◽  
Directed Mutagenesis ◽  
L Cells

We have investigated the role of the carbohydrate moiety on the HLA-B7 molecule in mAb and CTL recognition using oligonucleotide-directed mutagenesis and gene transfer techniques. A conservative substitution of asparagine to glutamine at amino acid 86 in HLA-B7 was created to abolish the unique glycosylation site present on all HLA molecules. A second mutant B7 molecule was made by substituting asparagine-aspartic acid-threonine for the resident lysine-aspartic acid/lysine tripeptide at amino acids 176-178, thus creating an N-linked glycan at amino acid 176, which is additionally present on all known murine H-2 class I antigens. Upon gene transfer into mouse and human cell recipients, the HLA-B7M176+ mutant and normal HLA-B7 expressed identical levels of surface protein. However, the binding of two mAbs (MB40.2 and MB40.3) thought to recognize different epitopes of the HLA-B7 molecule was completely eliminated. In contrast, the HLA-B7M86- mutant displayed no surface expression (mouse L cells) or minimal surface expression (human RD cells or mouse L cells coexpressing human beta 2 microglobulin [beta 2m]) after indirect immunofluorescence (IIF) and flow cytometric analysis with a panel of 12 HLA-B7 mAb reactive with monomorphic and polymorphic determinants. Immunoprecipitation analysis demonstrated that intracellular denatured mutant protein was present. Tunicamycin treatment did not rescue the expression of HLA-B7M86- antigens to the cell surface; while interferon did induce higher levels of surface expression. Tunicamycin treatment also did not allow binding of the mAbs MB40.2 or MB40.3 to HLA-B7M176+ mutant antigens, suggesting that the carbohydrate moiety itself was not directly involved in the recognition or conformation of these mAb epitopes. Further mutation of the B7M86- molecule to create a glycan moiety at amino acid position 176 (B7M86-/176+) did not rescue normal levels of surface expression. Finally, neither mutation was seen to affect recognition by a panel of 12 allospecific CTL clones. The low expression of HLA-B7M86- on the surface of human cell transfectants was sufficient to achieve lysis, albeit at a reduced efficiency, and lysis could be increased by interferon induction of higher levels of expression. Thus, the carbohydrate moiety on HLA antigens plays a minimal or nonexistent role in recognition by available mAb and allospecific CTL clones.

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 Gamma Irradiation of Aqueos Solution of L-Aspartic Acid, L-Aspartic Acid in Solid State, and L-Aspartic Acid Adsorbed into Na-Montmorillonite: Its Relevance in Chemistry Prebiotic

2021 ◽  
Vol 8 (2) ◽  
pp. 105-108
Author(s):  
A. Meléndez-López ◽  
M. F. García-Hurtado ◽  
J. Cruz-Castañeda ◽  
A. Negrón-Mendoza ◽  
S. Ramos-Bernal ◽  
...  
Keyword(s):  
Organic Matter ◽  
Amino Acid ◽  
Solid State ◽  
Aspartic Acid ◽  
Prebiotic Chemistry ◽  
High Energy ◽  
Energy Conditions ◽  
Gamma Radiolysis ◽  
The Stability

Aspartic acid is an amino acid present in the modern proteins, however, is considered a primitive amino acid hence its importance in prebiotic chemistry experiments studies. In some works of prebiotic chemistry have been studied the synthesis and the stability of organic matter under high energy sources, and the role of clays has been highlighted due to clays that can affect the reaction mechanisms in the radiolytic processes. The present work is focused on the study of the role of Namontmorillonite in the gamma radiolysis processes of L-aspartic acid. Gamma radiolysis processes were carried out in three different systems a) L-aspartic acid in aqueous solution; b) L-aspartic acid in solid-state; and c) L-aspartic acid adsorbed into Na-montmorillonite. L-aspartic acid was analyzed by high-performance liquid chromatography−electrospray ionization−mass spectrometry (HPLCESI-MS). The results showed that the decomposition of L-aspartic acid considerably decreased in the presence of clay thus highlighting the protector role of clays and favors the stability of organic matter even under the possible high energy conditions of primitive environments. The principal product ofgamma radiolysis of L-aspartic acid was succinic acid produced by deamination reaction. On the other hand, when aspartic acid was irradiated in solid-state the main product was the L-aspartic acid dimer. Both radiolysis products are important for chemical evolution processes for L-aspartic acid in primitive environments.

Sign in / Sign up

Export Citation Format

Share Document