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Literature summary for 3.5.2.6 extracted from

  • Kumar, G.; Biswal, S.; Nathan, S.; Ghosh, A.S.
    Glutamate residues at positions 162 and 164 influence the beta-lactamase activity of SHV-14 obtained from Klebsiella pneumoniae (2018), FEMS Microbiol. Lett., 365, fnx259 .
    View publication on PubMed

Cloned(Commentary)

Cloned (Comment) Organism
gene blaSHV-14, recombinant expression of N-terminally His6-tagged wild-type and mutant enzymes in Escherichia coli strains BL21(DE3) and CS109. On expression, the clone of wild-type SHV-14 aggravates the resistance of host by 60-500 folds against penicillin and cephalosporin groups of antibiotics, while the expression of mutated enzymes (especially E164A) substantially reduces the resistance level as compared to the wild-type, and the results are in synchrony with the estimated enzymatic efficiencies of wild-type and mutated proteins Klebsiella pneumoniae

Protein Variants

Protein Variants Comment Organism
E162A site-directed mutagenesis, mutation of the an omega loop glutamate, the mutant shows reduced activity against penicilin and cepahlosporin antibiotics in recombinant Escherichia coli cells and reduces the resistance level of the cells Klebsiella pneumoniae
E164A site-directed mutagenesis, mutation of the an omega loop glutamate, the mutant shows highly reduced activity against penicilin and cepahlosporin antibiotics in recombinant Escherichia coli cells and potently reduces the resistance level of the cells Klebsiella pneumoniae
E167A site-directed mutagenesis, mutation of the an omega loop glutamate, the mutant shows reduced activity against penicilin and cepahlosporin antibiotics in recombinant Escherichia coli cells and reduces the resistance level of the cells Klebsiella pneumoniae

Inhibitors

Inhibitors Comment Organism Structure
clavulanic acid
-
Klebsiella pneumoniae
additional information mutation at the glutamate residues in the omega-loop of SHV-14, i.e. E126, E164, and E167, render beta-lactamase inhibitors more efficient Klebsiella pneumoniae
Sulbactam
-
Klebsiella pneumoniae

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
additional information Michaelis-Menten steady-state kinetics studies Klebsiella pneumoniae
0.032
-
ampicillin pH 7.5, 25°C, recombinant His-tagged wild-type enzyme Klebsiella pneumoniae
0.038
-
nitrocefin pH 7.5, 25°C, recombinant His-tagged mutant E162A Klebsiella pneumoniae
0.0383
-
cefoperazone pH 7.5, 25°C, recombinant His-tagged mutant E162A Klebsiella pneumoniae
0.041
-
amoxicillin pH 7.5, 25°C, recombinant His-tagged mutant E164A Klebsiella pneumoniae
0.045
-
nitrocefin pH 7.5, 25°C, recombinant His-tagged wild-type enzyme Klebsiella pneumoniae
0.045
-
cephalothin pH 7.5, 25°C, recombinant His-tagged wild-type enzyme Klebsiella pneumoniae
0.05414
-
amoxicillin pH 7.5, 25°C, recombinant His-tagged mutant E162A Klebsiella pneumoniae
0.0557
-
piperacillin pH 7.5, 25°C, recombinant His-tagged mutant E162A Klebsiella pneumoniae
0.057
-
cephalothin pH 7.5, 25°C, recombinant His-tagged mutant E162A Klebsiella pneumoniae
0.0574
-
cefoperazone pH 7.5, 25°C, recombinant His-tagged wild-type enzyme Klebsiella pneumoniae
0.058
-
piperacillin pH 7.5, 25°C, recombinant His-tagged wild-type enzyme Klebsiella pneumoniae
0.0697
-
amoxicillin pH 7.5, 25°C, recombinant His-tagged wild-type enzyme Klebsiella pneumoniae
0.0716
-
piperacillin pH 7.5, 25°C, recombinant His-tagged mutant E164A Klebsiella pneumoniae
0.0962
-
cefoperazone pH 7.5, 25°C, recombinant His-tagged mutant E164A Klebsiella pneumoniae
0.1893
-
nitrocefin pH 7.5, 25°C, recombinant His-tagged mutant E164A Klebsiella pneumoniae
0.227
-
cephalothin pH 7.5, 25°C, recombinant His-tagged mutant E164A Klebsiella pneumoniae
0.414
-
ampicillin pH 7.5, 25°C, recombinant His-tagged mutant E162A Klebsiella pneumoniae
0.615
-
ampicillin pH 7.5, 25°C, recombinant His-tagged mutant E164A Klebsiella pneumoniae

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
amoxicillin + H2O Klebsiella pneumoniae
-
(2R,4S)-2-[(R)-[[(2R)-2-amino-2-(4-hydroxyphenyl)acetyl]amino](carboxy)methyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxylic acid
-
?
amoxicillin + H2O Klebsiella pneumoniae MTCC 3384
-
(2R,4S)-2-[(R)-[[(2R)-2-amino-2-(4-hydroxyphenyl)acetyl]amino](carboxy)methyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxylic acid
-
?
ampicillin + H2O Klebsiella pneumoniae
-
(2R,4S)-2-[(R)-[[(2R)-2-amino-2-phenylacetyl]amino](carboxy)methyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxylic acid
-
?
ampicillin + H2O Klebsiella pneumoniae MTCC 3384
-
(2R,4S)-2-[(R)-[[(2R)-2-amino-2-phenylacetyl]amino](carboxy)methyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxylic acid
-
?
cefoperazone + H2O Klebsiella pneumoniae
-
(2R)-2-[(R)-carboxy([(2S)-2-[(4-ethyl-2,3-dioxopiperazine-1-carbonyl)amino]-2-(4-hydroxyphenyl)acetyl]amino)methyl]-5-([(1-methyl-1H-tetrazol-5-yl)sulfanyl]methyl)-3,6-dihydro-2H-1,3-thiazine-4-carboxylic acid
-
?
cefotaxime + H2O Klebsiella pneumoniae
-
(2R)-5-[(acetyloxy)methyl]-2-[(R)-[[(2E)-2-(2-amino-1,3-thiazol-4-yl)-2-(methoxyimino)acetyl]amino](carboxy)methyl]-3,6-dihydro-2H-1,3-thiazine-4-carboxylic acid
-
?
cefotaxime + H2O Klebsiella pneumoniae MTCC 3384
-
(2R)-5-[(acetyloxy)methyl]-2-[(R)-[[(2E)-2-(2-amino-1,3-thiazol-4-yl)-2-(methoxyimino)acetyl]amino](carboxy)methyl]-3,6-dihydro-2H-1,3-thiazine-4-carboxylic acid
-
?
ceftazidime + H2O Klebsiella pneumoniae
-
?
-
?
cephalothin + H2O Klebsiella pneumoniae
-
(2R)-5-[(acetyloxy)methyl]-2-[(R)-carboxy[2-(thiophen-2-yl)acetamido]methyl]-3,6-dihydro-2H-1,3-thiazine-4-carboxylic acid
-
?
imipenem + H2O Klebsiella pneumoniae
-
(5R)-3-[[2-(carbonoimidoylamino)ethyl]sulfanyl]-5-[(1S,2R)-1-carboxy-2-hydroxypropyl]-4,5-dihydro-1H-pyrrole-2-carboxylic acid
-
?
meropenem + H2O Klebsiella pneumoniae
-
(4R,5S)-5-[(1S,2R)-1-carboxy-2-hydroxypropyl]-3-[[(3S,5S)-5-(dimethylcarbamoyl)pyrrolidin-3-yl]sulfanyl]-4-methyl-4,5-dihydro-1H-pyrrole-2-carboxylic acid
-
?
additional information Klebsiella pneumoniae antibiotic sensitivity of recombinant wild-type and mutant blaSHV-14, overview ?
-
-
additional information Klebsiella pneumoniae MTCC 3384 antibiotic sensitivity of recombinant wild-type and mutant blaSHV-14, overview ?
-
-
nitrocefin + H2O Klebsiella pneumoniae
-
(2R)-2-((R)-carboxy[2-(thiophen-2-yl)acetamido]methyl)-5-[(E)-2-(2,4-dinitrophenyl)ethenyl]-3,6-dihydro-2H-1,3-thiazine-4-carboxylic acid
-
?
penicillin G + H2O Klebsiella pneumoniae
-
(2R,4S)-2-[(R)-carboxy[(phenylacetyl)amino]methyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxylic acid
-
?
penicillin G + H2O Klebsiella pneumoniae MTCC 3384
-
(2R,4S)-2-[(R)-carboxy[(phenylacetyl)amino]methyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxylic acid
-
?
piperacillin + H2O Klebsiella pneumoniae
-
(2R,4S)-2-[(R)-carboxy([(2R)-2-[(4-ethyl-2,3-dioxopiperazine-1-carbonyl)amino]-2-phenylacetyl]amino)methyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxylic acid
-
?

Organism

Organism UniProt Comment Textmining
Klebsiella pneumoniae Q9F918
-
-
Klebsiella pneumoniae MTCC 3384 Q9F918
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant N-terminally His6-tagged wild-type and mutant enzymes from Escherichia coli by nickel affinity chromatography Klebsiella pneumoniae

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
amoxicillin + H2O
-
Klebsiella pneumoniae (2R,4S)-2-[(R)-[[(2R)-2-amino-2-(4-hydroxyphenyl)acetyl]amino](carboxy)methyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxylic acid
-
?
amoxicillin + H2O
-
Klebsiella pneumoniae MTCC 3384 (2R,4S)-2-[(R)-[[(2R)-2-amino-2-(4-hydroxyphenyl)acetyl]amino](carboxy)methyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxylic acid
-
?
ampicilin + H2O
-
Klebsiella pneumoniae (2R,4S)-2-[(R)-[[(2R)-2-amino-2-phenylacetyl]amino](carboxy)methyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxylic acid
-
?
ampicillin + H2O
-
Klebsiella pneumoniae (2R,4S)-2-[(R)-[[(2R)-2-amino-2-phenylacetyl]amino](carboxy)methyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxylic acid
-
?
ampicillin + H2O
-
Klebsiella pneumoniae MTCC 3384 (2R,4S)-2-[(R)-[[(2R)-2-amino-2-phenylacetyl]amino](carboxy)methyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxylic acid
-
?
cefoperazone + H2O
-
Klebsiella pneumoniae (2R)-2-[(R)-carboxy([(2S)-2-[(4-ethyl-2,3-dioxopiperazine-1-carbonyl)amino]-2-(4-hydroxyphenyl)acetyl]amino)methyl]-5-([(1-methyl-1H-tetrazol-5-yl)sulfanyl]methyl)-3,6-dihydro-2H-1,3-thiazine-4-carboxylic acid
-
?
cefotaxime + H2O
-
Klebsiella pneumoniae (2R)-5-[(acetyloxy)methyl]-2-[(R)-[[(2E)-2-(2-amino-1,3-thiazol-4-yl)-2-(methoxyimino)acetyl]amino](carboxy)methyl]-3,6-dihydro-2H-1,3-thiazine-4-carboxylic acid
-
?
cefotaxime + H2O
-
Klebsiella pneumoniae MTCC 3384 (2R)-5-[(acetyloxy)methyl]-2-[(R)-[[(2E)-2-(2-amino-1,3-thiazol-4-yl)-2-(methoxyimino)acetyl]amino](carboxy)methyl]-3,6-dihydro-2H-1,3-thiazine-4-carboxylic acid
-
?
ceftazidime + H2O
-
Klebsiella pneumoniae ?
-
?
cephalothin + H2O
-
Klebsiella pneumoniae (2R)-5-[(acetyloxy)methyl]-2-[(R)-carboxy[2-(thiophen-2-yl)acetamido]methyl]-3,6-dihydro-2H-1,3-thiazine-4-carboxylic acid
-
?
imipenem + H2O
-
Klebsiella pneumoniae (5R)-3-[[2-(carbonoimidoylamino)ethyl]sulfanyl]-5-[(1S,2R)-1-carboxy-2-hydroxypropyl]-4,5-dihydro-1H-pyrrole-2-carboxylic acid
-
?
meropenem + H2O
-
Klebsiella pneumoniae (4R,5S)-5-[(1S,2R)-1-carboxy-2-hydroxypropyl]-3-[[(3S,5S)-5-(dimethylcarbamoyl)pyrrolidin-3-yl]sulfanyl]-4-methyl-4,5-dihydro-1H-pyrrole-2-carboxylic acid
-
?
additional information antibiotic sensitivity of recombinant wild-type and mutant blaSHV-14, overview Klebsiella pneumoniae ?
-
-
additional information antibiotic sensitivity of recombinant wild-type and mutant blaSHV-14, overview Klebsiella pneumoniae MTCC 3384 ?
-
-
nitrocefin + H2O
-
Klebsiella pneumoniae (2R)-2-((R)-carboxy[2-(thiophen-2-yl)acetamido]methyl)-5-[(E)-2-(2,4-dinitrophenyl)ethenyl]-3,6-dihydro-2H-1,3-thiazine-4-carboxylic acid
-
?
penicillin G + H2O
-
Klebsiella pneumoniae (2R,4S)-2-[(R)-carboxy[(phenylacetyl)amino]methyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxylic acid
-
?
penicillin G + H2O
-
Klebsiella pneumoniae MTCC 3384 (2R,4S)-2-[(R)-carboxy[(phenylacetyl)amino]methyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxylic acid
-
?
piperacillin + H2O
-
Klebsiella pneumoniae (2R,4S)-2-[(R)-carboxy([(2R)-2-[(4-ethyl-2,3-dioxopiperazine-1-carbonyl)amino]-2-phenylacetyl]amino)methyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxylic acid
-
?

Subunits

Subunits Comment Organism
? x * 30000, about, recombinant wild-type and mutant enzymes, SDS-PAGE and MALDI-ToF MS analysis Klebsiella pneumoniae

Synonyms

Synonyms Comment Organism
blaSHV-14
-
Klebsiella pneumoniae
SHV-14 beta-lactamase
-
Klebsiella pneumoniae

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
25
-
assay at Klebsiella pneumoniae

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.08
-
nitrocefin pH 7.5, 25°C, recombinant His-tagged mutant E162A Klebsiella pneumoniae
0.125
-
nitrocefin pH 7.5, 25°C, recombinant His-tagged mutant E164A Klebsiella pneumoniae
0.21
-
cefoperazone pH 7.5, 25°C, recombinant His-tagged mutant E162A Klebsiella pneumoniae
0.43
-
cephalothin pH 7.5, 25°C, recombinant His-tagged mutant E164A Klebsiella pneumoniae
0.44
-
cephalothin pH 7.5, 25°C, recombinant His-tagged mutant E162A Klebsiella pneumoniae
0.705
-
cefoperazone pH 7.5, 25°C, recombinant His-tagged mutant E164A Klebsiella pneumoniae
2.03
-
amoxicillin pH 7.5, 25°C, recombinant His-tagged mutant E164A Klebsiella pneumoniae
2.2
-
piperacillin pH 7.5, 25°C, recombinant His-tagged mutant E164A Klebsiella pneumoniae
2.38
-
amoxicillin pH 7.5, 25°C, recombinant His-tagged mutant E162A Klebsiella pneumoniae
2.46
-
piperacillin pH 7.5, 25°C, recombinant His-tagged mutant E162A Klebsiella pneumoniae
5.85
-
nitrocefin pH 7.5, 25°C, recombinant His-tagged wild-type enzyme Klebsiella pneumoniae
15
-
ampicillin pH 7.5, 25°C, recombinant His-tagged mutant E162A Klebsiella pneumoniae
21.5
-
ampicillin pH 7.5, 25°C, recombinant His-tagged mutant E164A Klebsiella pneumoniae
113
-
cephalothin pH 7.5, 25°C, recombinant His-tagged wild-type enzyme Klebsiella pneumoniae
229.5
-
cefoperazone pH 7.5, 25°C, recombinant His-tagged wild-type enzyme Klebsiella pneumoniae
532.5
-
amoxicillin pH 7.5, 25°C, recombinant His-tagged wild-type enzyme Klebsiella pneumoniae
544
-
ampicillin pH 7.5, 25°C, recombinant His-tagged wild-type enzyme Klebsiella pneumoniae
677
-
piperacillin pH 7.5, 25°C, recombinant His-tagged wild-type enzyme Klebsiella pneumoniae

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.5
-
assay at Klebsiella pneumoniae

General Information

General Information Comment Organism
malfunction mutation at the glutamate residues in the omega-loop of SHV-14, i.e. E126, E164, and E167, can considerably modulate the beta-lactam sensitivity and reduce hydrolysis. The mutations in SHV-14 render beta-lactamase inhibitors more efficient. The mutations alter the active site configuration of SHV-14 Klebsiella pneumoniae
additional information substrate binding to active site residues of SHV-14 wild-type and mutant enzyme proteins, structures, overview Klebsiella pneumoniae
physiological function extensive production of SHV-14 beta-lactamase makes Klebsiella pneumoniae resistant to beta-lactams. The presence of an omega-loop has been reported to influence the beta-lactamase activity, which is also present in SHV-14 Klebsiella pneumoniae

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
0.21
-
nitrocefin pH 7.5, 25°C, recombinant His-tagged mutant E162A Klebsiella pneumoniae
0.66
-
nitrocefin pH 7.5, 25°C, recombinant His-tagged mutant E164A Klebsiella pneumoniae
1.89
-
cephalothin pH 7.5, 25°C, recombinant His-tagged mutant E164A Klebsiella pneumoniae
5.48
-
cefoperazone pH 7.5, 25°C, recombinant His-tagged mutant E162A Klebsiella pneumoniae
7.33
-
cefoperazone pH 7.5, 25°C, recombinant His-tagged mutant E164A Klebsiella pneumoniae
7.72
-
cephalothin pH 7.5, 25°C, recombinant His-tagged mutant E162A Klebsiella pneumoniae
30.73
-
piperacillin pH 7.5, 25°C, recombinant His-tagged mutant E164A Klebsiella pneumoniae
34.96
-
ampicillin pH 7.5, 25°C, recombinant His-tagged mutant E164A Klebsiella pneumoniae
36.23
-
ampicillin pH 7.5, 25°C, recombinant His-tagged mutant E162A Klebsiella pneumoniae
43.96
-
amoxicillin pH 7.5, 25°C, recombinant His-tagged mutant E162A Klebsiella pneumoniae
44.17
-
piperacillin pH 7.5, 25°C, recombinant His-tagged mutant E162A Klebsiella pneumoniae
49.51
-
amoxicillin pH 7.5, 25°C, recombinant His-tagged mutant E164A Klebsiella pneumoniae
130
-
nitrocefin pH 7.5, 25°C, recombinant His-tagged wild-type enzyme Klebsiella pneumoniae
2511.1
-
cephalothin pH 7.5, 25°C, recombinant His-tagged wild-type enzyme Klebsiella pneumoniae
3998.3
-
cefoperazone pH 7.5, 25°C, recombinant His-tagged wild-type enzyme Klebsiella pneumoniae
11672.4
-
piperacillin pH 7.5, 25°C, recombinant His-tagged wild-type enzyme Klebsiella pneumoniae
17000
-
ampicillin pH 7.5, 25°C, recombinant His-tagged wild-type enzyme Klebsiella pneumoniae
36334.3
-
amoxicillin pH 7.5, 25°C, recombinant His-tagged wild-type enzyme Klebsiella pneumoniae