scholarly journals Identification of Arg-12 in the active site of Escherichia coli K1 CMP-sialic acid synthetase

1999 ◽  
Vol 343 (2) ◽  
pp. 397-402 ◽  
Author(s):  
Daniel M. STOUGHTON ◽  
Gerardo ZAPATA ◽  
Robert PICONE ◽  
Willie F. VANN
Keyword(s):  
Escherichia Coli ◽  
Sialic Acid ◽  
Active Site ◽  
Positive Charge ◽  
Enzymic Activity ◽  
Site Directed Mutagenesis ◽  
E Coli ◽  
Steady State Kinetic ◽  
State Kinetic ◽  
Escherichia Coli K1

Escherichia coli K1 CMP-sialic acid synthetase catalyses the synthesis of CMP-sialic acid from CTP and sialic acid. The active site of the 418 amino acid E. coli enzyme was localized to its N-terminal half. The bacterial CMP-sialic acid synthetase enzymes have a conserved motif, IAIIPARXXSKGLXXKN, at their N-termini. Several basic residues have been identified at or near the active site of the E. coli enzyme by chemical modification and site-directed mutagenesis. Only one of the lysines in the N-terminal motif, Lys-21, appears to be essential for activity. Mutation of Lys-21 in the N-terminal motif results in an inactive enzyme. Furthermore, Arg-12 of the N-terminal motif appears to be an active-site residue, based on the following evidence. Substituting Arg-12 with glycine or alanine resulted in inactive enzymes, indicating that this residue is required for enzymic activity. The Arg-12 → Lys mutant was partially active, demonstrating that a positive charge is required at this site. Steady-state kinetic analysis reveals changes in kcat, Km and Ks for CTP, which implicates Arg-12 in catalysis and substrate binding.

scholarly journals Identification of Arg-12 in the active site of Escherichia coli K1 CMP-sialic acid synthetase

1999 ◽  
Vol 343 (2) ◽  
pp. 397 ◽  
Author(s):  
Daniel M. STOUGHTON ◽  
Gerardo ZAPATA ◽  
Robert PICONE ◽  
Willie F. VANN
Keyword(s):  
Escherichia Coli ◽  
Sialic Acid ◽  
Active Site ◽  
Escherichia Coli K1

scholarly journals Sequences near the Active Site in Chimeric Penicillin Binding Proteins 5 and 6 Affect Uniform Morphology of Escherichia coli

2003 ◽  
Vol 185 (7) ◽  
pp. 2178-2186 ◽  
Author(s):  
Anindya S. Ghosh ◽  
Kevin D. Young
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Active Site ◽  
Site Directed Mutagenesis ◽  
Globular Domain ◽  
Morphological Effect ◽  
E Coli ◽  
Amino Terminal ◽  
Terminal Amino ◽  
Domain I

ABSTRACT Penicillin binding protein (PBP) 5, a dd-carboxypeptidase that removes the terminal d-alanine from peptide side chains of peptidoglycan, plays an important role in creating and maintaining the uniform cell shape of Escherichia coli. PBP 6, a highly similar homologue, cannot substitute for PBP 5 in this respect. Previously, we localized the shape-maintaining characteristics of PBP 5 to the globular domain that contains the active site (domain I), where PBPs 5 and 6 share substantial identity. To identify the specific segment of domain I responsible for shape control, we created a set of hybrids and determined which ones complemented the aberrant morphology of a misshapen PBP mutant, E. coli CS703-1. Fusion proteins were constructed in which 47, 199 and 228 amino-terminal amino acids of one PBP were fused to the corresponding carboxy-terminal amino acids of the other. The morphological phenotype was reversed only by hybrid proteins containing PBP 5 residues 200 to 228, which are located next to the KTG motif of the active site. Because residues 220 to 228 were identical in these proteins, the morphological effect was determined by alterations in amino acids 200 to 219. To confirm the importance of this segment, we constructed mosaic proteins in which these 20 amino acids were grafted from PBP 5 into PBP 6 and vice versa. The PBP 6/5/6 mosaic complemented the aberrant morphology of CS703-1, whereas PBP 5/6/5 did not. Site-directed mutagenesis demonstrated that the Asp218 and Lys219 residues were important for shape maintenance by these mosaic PBPs, but the same mutations in wild-type PBP 5 did not eliminate its shape-promoting activity. Homologous enzymes from five other bacteria also complemented the phenotype of CS703-1. The overall conclusion is that creation of a bacterial cell of regular diameter and uniform contour apparently depends primarily on a slight alteration of the enzymatic activity or substrate accessibility at the active site of E. coli PBP 5.

scholarly journals Identification of amino acid residues at the active site of endosialidase that dissociate the polysialic acid binding and cleaving activities in Escherichia coli K1 bacteriophages

2007 ◽  
Vol 405 (3) ◽  
pp. 465-472 ◽  
Author(s):  
Elina Jakobsson ◽  
Anne Jokilammi ◽  
Juha Aalto ◽  
Pauli Ollikka ◽  
Jukka V. Lehtonen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Active Site ◽  
Structural Model ◽  
Polysialic Acid ◽  
Binding Activity ◽  
Enzymic Activity ◽  
Amino Acid Residues ◽  
Post Translational Modification ◽  
Escherichia Coli K1

Endosialidase (endo-N-acetylneuraminidase) is a tailspike enzyme of bacteriophages specific for human pathogenic Escherichia coli K1, which specifically recognizes and degrades polySia (polysialic acid). polySia is also a polysaccharide of the capsules of other meningitis- and sepsis-causing bacteria, and a post-translational modification of the NCAM (neural cell-adhesion molecule). We have cloned and sequenced three spontaneously mutated endosialidases of the PK1A bacteriophage and one of the PK1E bacteriophage which display lost or residual enzyme activity but retain the binding activity to polySia. Single to triple amino acid substitutions were identified, and back-mutation constructs indicated that single substitutions accounted for only partial reduction of enzymic activity. A homology-based structural model of endosialidase revealed that all substituted amino acid residues localize to the active site of the enzyme. The results reveal the importance of non-catalytic amino acid residues for the enzymatic activity. The results reveal the molecular background for the dissociation of the polySia binding and cleaving activities of endosialidase and for the evolvement of ‘host range’ mutants of E. coli K1 bacteriophages.

scholarly journals The effect of replacing the conserved active-site residues His-264, Asp-312 and Arg-314 on the binding and catalytic properties of Escherichia coli citrate synthase

1994 ◽  
Vol 300 (3) ◽  
pp. 765-770 ◽  
Author(s):  
W J Man ◽  
Y Li ◽  
C D O'Connor ◽  
D C Wilton
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Citrate Synthase ◽  
Salt Bridge ◽  
Site Directed Mutagenesis ◽  
Active Site Residues ◽  
E Coli ◽  
Rate Limiting Step ◽  
Rate Limiting

The first step in the overall catalytic mechanism of citrate synthase is the binding and polarization of oxaloacetate. Active-site residues Arg-314, Asp-312 and His-264 in Escherichia coli citrate synthase, which are involved in oxaloacetate binding, were converted by site-directed mutagenesis to Gln-314, Asn-312 and Asn-264 respectively. The R314Q and D312N mutants expressed negligible overall catalytic activity at pH 8.0, the normal assay pH, but substantial activities for the partial reactions that reflect the cleavage and hydrolysis of the substrate intermediate citryl-CoA. However, when the pH was lowered to 7.0, the overall reaction of the mutants became significant, in contrast to the wild-type enzyme, whereas the two mutants exhibited reduced activities for the partial reactions. This result is consistent with the existence of a rate-limiting step between the two partial reactions for these mutants that is pH-dependent. The Km for oxaloacetate for the two mutants was increased 10-fold and was paralleled by an increase in the Km for citryl-CoA, whereas the Km for acetyl-CoA was increased only 2-fold. Overall, there was a striking parallel between the results obtained for these two mutants, which suggests that they are functionally linked in the E. coli enzyme. The equivalent of these two residues form a salt bridge in the pig heart citrate synthase crystal structure. The H264N mutant, in which the amide nitrogen of asparagine should mimic the delta-nitrogen of histidine, showed negligible activity in terms of both overall and partial catalysis, which may result from a hindrance of conformational change upon oxaloacetate binding. The affinity of this mutant for oxaloacetate appeared to be greatly reduced when investigated using indirect fluorescence and chemical modification techniques.

scholarly journals Proton-linked l-fucose transport in Escherichia coli

1987 ◽  
Vol 248 (2) ◽  
pp. 495-500 ◽  
Author(s):  
S A Bradley ◽  
C R Tinsley ◽  
J A R Muiry ◽  
P J F Henderson
Keyword(s):  
Escherichia Coli ◽  
Alkaline Ph ◽  
Electrochemical Gradient ◽  
Ph Change ◽  
Ph Optimum ◽  
Kinetic Measurements ◽  
E Coli ◽  
Steady State Kinetic ◽  
Periplasmic Proteins ◽  
State Kinetic

1. Addition of L-fucose to energy-depleted anaerobic suspensions of Escherichia coli elicited an uncoupler-sensitive alkaline pH change diagnostic of L-fucose/H+ symport activity. 2. L-Galactose or D-arabinose were also substrates, but not inducers, for the L-fucose/H+ symporter. 3. L-Fucose transport into subcellular vesicles was dependent upon respiration, displayed a pH optimum of about 5.5, and was inhibited by protonophores and ionophores. 4. These results showed that L-fucose transport into E. coli was energized by the transmembrane electrochemical gradient of protons. 5. Neither steady state kinetic measurements nor assays of L-fucose binding to periplasmic proteins revealed the existence of a second L-fucose transport system.

scholarly journals A genetically detoxified derivative of heat-labile Escherichia coli enterotoxin induces neutralizing antibodies against the A subunit.

1994 ◽  
Vol 180 (6) ◽  
pp. 2147-2153 ◽  
Author(s):  
M Pizza ◽  
M R Fontana ◽  
M M Giuliani ◽  
M Domenighini ◽  
C Magagnoli ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cholera Toxin ◽  
Neutralizing Antibodies ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
E Coli ◽  
Heat Labile ◽  
A Subunit ◽  
Adp Ribosyltransferase ◽  
Derivatives Of

Escherichia coli enterotoxin (LT) and the homologous cholera toxin (CT) are A-B toxins that cause travelers' diarrhea and cholera, respectively. So far, experimental live and killed vaccines against these diseases have been developed using only the nontoxic B portion of these toxins. The enzymatically active A subunit has not been used because it is responsible for the toxicity and it is reported to induce a negligible titer of toxin neutralizing antibodies. We used site-directed mutagenesis to inactivate the ADP-ribosyltransferase activity of the A subunit and obtained nontoxic derivatives of LT that elicited a good titer of neutralizing antibodies recognizing the A subunit. These LT mutants and equivalent mutants of CT may be used to improve live and killed vaccines against cholera and enterotoxinogenic E. coli.

scholarly journals Genome-Wide Screening Identifies Six Genes That Are Associated with Susceptibility to Escherichia coli Microcin PDI

2015 ◽  
Vol 81 (20) ◽  
pp. 6953-6963 ◽  
Author(s):  
Zhe Zhao ◽  
Lauren J. Eberhart ◽  
Lisa H. Orfe ◽  
Shao-Yeh Lu ◽  
Thomas E. Besser ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Atp Synthase ◽  
Disulfide Bonds ◽  
Gene Knockout ◽  
Single Gene ◽  
Site Directed Mutagenesis ◽  
Content Type ◽  
E Coli ◽  
Synthase Inhibitor ◽  
Loss Of Susceptibility

ABSTRACTThe microcin PDI inhibits a diverse group of pathogenicEscherichia colistrains. Coculture of a single-gene knockout library (BW25113;n= 3,985 mutants) against a microcin PDI-producing strain (E. coli25) identified six mutants that were not susceptible (ΔatpA, ΔatpF, ΔdsbA, ΔdsbB, ΔompF, and ΔompR). Complementation of these genes restored susceptibility in all cases, and the loss of susceptibility was confirmed through independent gene knockouts inE. coliO157:H7 Sakai. Heterologous expression ofE. coliompFconferred susceptibility toSalmonella entericaandYersinia enterocoliticastrains that are normally unaffected by microcin PDI. The expression of chimeric OmpF and site-directed mutagenesis revealed that the K47G48N49region within the first extracellular loop ofE. coliOmpF is a putative binding site for microcin PDI. OmpR is a transcriptional regulator forompF, and consequently loss of susceptibility by the ΔompRstrain most likely is related to this function. Deletion of AtpA and AtpF, as well as AtpE and AtpH (missed in the original library screen), resulted in the loss of susceptibility to microcin PDI and the loss of ATP synthase function. Coculture of a susceptible strain in the presence of an ATP synthase inhibitor resulted in a loss of susceptibility, confirming that a functional ATP synthase complex is required for microcin PDI activity. Intransexpression ofompFin the ΔdsbAand ΔdsbBstrains did not restore a susceptible phenotype, indicating that these proteins are probably involved with the formation of disulfide bonds for OmpF or microcin PDI.

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