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.

Amino acid residues in asparaginase (Escherichia coli Hap) associated with its enzymic activity

1971 ◽  
Vol 227 (1) ◽  
pp. 171-179 ◽  
Author(s):  
Yoshihisa Nishimura ◽  
Hiroshi Makino ◽  
Osamu Takenaka ◽  
Yuji Inada
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Enzymic Activity ◽  
Amino Acid Residues

scholarly journals Purification and characterization of human H-ras proteins expressed in Escherichia coli.

1985 ◽  
Vol 5 (5) ◽  
pp. 1015-1024 ◽  
Author(s):  
M Gross ◽  
R W Sweet ◽  
G Sathe ◽  
S Yokoyama ◽  
O Fasano ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Binding Activity ◽  
Full Length ◽  
Binding Kinetics ◽  
Amino Acid Residues ◽  
Ras Proteins ◽  
Truncated Protein ◽  
Amino Terminal ◽  
Carboxyl Terminal

The full-length normal and T24 mutant human H-ras proteins and two truncated derivatives of the T24 mutant were expressed efficiently in Escherichia coli. The proteins accumulated to 1 to 5% of total cellular protein, and each was specifically recognized by anti-ras monoclonal antibodies. The two full-length proteins as well as a carboxyl-terminal truncated derivative (deleted for 23 amino acid residues) were soluble upon cell lysis and were purified to 90% homogeneity without the use of denaturants. In contrast, an amino-terminal truncated ras derivative (deleted for 22 amino acid residues) required treatment with urea for its solubilization. The guanine nucleotide binding activity of these four proteins was assessed by a combination of ligand binding on proteins blots, immunoprecipitation, and standard filter binding procedures. The full-length proteins showed similar binding kinetics and a stoichiometry approaching 1 mol of GTP bound per mol of protein. The showed similar binding kinetics and a stoichiometry approaching 1 mol of GTP bound per mol of protein. The carboxyl-terminal truncated protein also bound GTP, but to a reduced extent, whereas the amino-terminal truncated protein did not have binding activity. Apparently, the carboxyl-terminal domain of ras, although important for transforming function, does not play a critical role in GTP binding.

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

1999 ◽  
Vol 343 (2) ◽  
pp. 397-402 ◽  
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 Functional groups in the activity and regulation of Escherichia coli citrate synthase

1973 ◽  
Vol 135 (3) ◽  
pp. 513-524 ◽  
Author(s):  
Michael J. Danson ◽  
P. David J. Weitzman
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Citrate Synthase ◽  
Ph Dependence ◽  
Enzymic Activity ◽  
Amino Acid Residues ◽  
Specific Chemical ◽  
Photo Oxidation ◽  
Chemical Reagents ◽  
Effector Site

1. Citrate synthase has been purified from Escherichia coli and shown to exist at an equilibrium between three forms: monomer (mol.wt. 57000), tetramer (mol.wt. 230000) and, possibly, octamer. Modification of the enzyme by photo-oxidation and by treatment with specific chemical reagents has been carried out to gain information on the amino acid residues involved in enzymic activity and in the inhibition of activity by NADH and α-oxoglutarate. 2. Several photo-oxidizable amino acids appear to be involved in activity. The nature of the pH-dependence of their rates of photo-oxidation with Methylene Blue suggests that these are histidines, a conclusion supported by the greater rate of photo-inactivation with Rose Bengal and the destruction of activity by diethyl pyrocarbonate. 3. The participation of histidine at the α-oxoglutarate effector site is indicated by photo-oxidation and the participation of cysteine at the NADH effector site suggested by photo-oxidation is confirmed by the desensitization to NADH produced by treatment with 5,5′-dithiobis-(2-nitrobenzoate). Inactivation of the enzyme after modification with this reagent suggests the additional involvement of cysteine in catalytic activity. 4. Amino acid analyses of native and photo-oxidized enzyme are consistent with these conclusions. 5. Modification with 2-hydroxy-5-nitrobenzyl bromide indicates the participation of tryptophan in the activity of the enzyme.

scholarly journals Purification and characterization of human H-ras proteins expressed in Escherichia coli

1985 ◽  
Vol 5 (5) ◽  
pp. 1015-1024
Author(s):  
M Gross ◽  
R W Sweet ◽  
G Sathe ◽  
S Yokoyama ◽  
O Fasano ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Binding Activity ◽  
Full Length ◽  
Binding Kinetics ◽  
Amino Acid Residues ◽  
Ras Proteins ◽  
Truncated Protein ◽  
Amino Terminal ◽  
Carboxyl Terminal

The full-length normal and T24 mutant human H-ras proteins and two truncated derivatives of the T24 mutant were expressed efficiently in Escherichia coli. The proteins accumulated to 1 to 5% of total cellular protein, and each was specifically recognized by anti-ras monoclonal antibodies. The two full-length proteins as well as a carboxyl-terminal truncated derivative (deleted for 23 amino acid residues) were soluble upon cell lysis and were purified to 90% homogeneity without the use of denaturants. In contrast, an amino-terminal truncated ras derivative (deleted for 22 amino acid residues) required treatment with urea for its solubilization. The guanine nucleotide binding activity of these four proteins was assessed by a combination of ligand binding on proteins blots, immunoprecipitation, and standard filter binding procedures. The full-length proteins showed similar binding kinetics and a stoichiometry approaching 1 mol of GTP bound per mol of protein. The showed similar binding kinetics and a stoichiometry approaching 1 mol of GTP bound per mol of protein. The carboxyl-terminal truncated protein also bound GTP, but to a reduced extent, whereas the amino-terminal truncated protein did not have binding activity. Apparently, the carboxyl-terminal domain of ras, although important for transforming function, does not play a critical role in GTP binding.

scholarly journals Derived amino acid sequence and identification of active site residues of Escherichia coli beta-hydroxydecanoyl thioester dehydrase.

1988 ◽  
Vol 263 (10) ◽  
pp. 4641-4646 ◽  
Author(s):  
J E Cronan ◽  
W B Li ◽  
R Coleman ◽  
M Narasimhan ◽  
D de Mendoza ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Active Site ◽  
Active Site Residues
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