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Literature summary for 3.5.2.6 extracted from

  • De Wals, P.Y.; Doucet, N.; Pelletier, J.N.
    High tolerance to simultaneous active-site mutations in TEM-1 beta-lactamase: Distinct mutational paths provide more generalized beta-lactam recognition (2009), Protein Sci., 18, 147-160.
    View publication on PubMedView publication on EuropePMC
Search for more literature for 3.5.2.6

Protein Variants

Protein Variants Comment Organism
G240H kcat/Km for cefotaxime is 5fold higher than wild-type value, kcat/Km for cephalothin is 6.4fold higher than wild-type value, kcat/Km for cefazolin is 2.3fold higher than wild-type value, kcat/Km for benzylpenicillin is 3.9fold lower than wild-type value plasmid pQE32Chl17
additional information investigation of the potential for increased recognition of an extended-spectrum cephalosporin by targeted sequence variation in TEM-1 plasmid pQE32Chl17

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.023
-
 cephalothin pH 7.0, 22°C, mutant enzyme G240H plasmid pQE32Chl17
0.062
-
 benzylpenicillin pH 7.0, 22°C, wild-type enzyme plasmid pQE32Chl17
0.095
-
 cefazolin pH 7.0, 22°C, mutant enzyme G240H plasmid pQE32Chl17
0.13
-
 cefazolin pH 7.0, 22°C, wild-type enzyme plasmid pQE32Chl17
0.18
-
 cephalothin pH 7.0, 22°C, wild-type enzyme plasmid pQE32Chl17
0.42
-
 benzylpenicillin pH 7.0, 22°C, mutant enzyme G240H plasmid pQE32Chl17
0.62
-
 cefotaxime pH 7.0, 22°C, mutant enzyme G240H plasmid pQE32Chl17
0.84
-
 cefotaxime pH 7.0, 22°C, wild-type enzyme plasmid pQE32Chl17

Organism

Organism UniProt Comment Textmining
plasmid pQE32Chl17
-
 the chloramphenicol acetyltransferase gene replaces the blaTEM-1 gene as the selectable marker, allowing plasmid maintenance using 0.0125 mg/ml chloramphenicol. The blaTEM-1 gene is then subcloned under control of the IPTG-inducible promoter and mutated. The resulting constructs harbor a 6-histidine tag that is N-terminal to the native TEM-1 signal peptide. Upon processing of the signal peptide during expression and maturation in Escherichia coli, the histidine tag is also removed
-

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
benzylpenicillin + H2O
-
 plasmid pQE32Chl17 (2R,4S)-2-[(R)-carboxy[(phenylacetyl)amino]methyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxylic acid
-
 ?
cefazolin + H2O
-
 plasmid pQE32Chl17 (2R)-2-[(R)-carboxy[(1H-tetrazol-1-ylacetyl)amino]methyl]-5-[[(5-methyl-1,3,4-thiadiazol-2-yl)sulfanyl]methyl]-3,6-dihydro-2H-1,3-thiazine-4-carboxylic acid
-
 ?
cefotaxime + H2O
-
 plasmid pQE32Chl17 (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
-
 ?
cephalothin + H2O
-
 plasmid pQE32Chl17 (2R)-5-[(acetyloxy)methyl]-2-[(R)-carboxy[(thiophen-2-ylacetyl)amino]methyl]-3,6-dihydro-2H-1,3-thiazine-4-carboxylic acid
-
 ?

Synonyms

Synonyms Comment Organism
TEM-1
-
 plasmid pQE32Chl17

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
22
-
 assay at plasmid pQE32Chl17

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.74
-
 cefotaxime pH 7.0, 22°C, wild-type enzyme plasmid pQE32Chl17
2.7
-
 cefotaxime pH 7.0, 22°C, mutant enzyme G240H plasmid pQE32Chl17
63
-
 cephalothin pH 7.0, 22°C, mutant enzyme G240H plasmid pQE32Chl17
69
-
 cefazolin pH 7.0, 22°C, wild-type enzyme plasmid pQE32Chl17
77
-
 cephalothin pH 7.0, 22°C, wild-type enzyme plasmid pQE32Chl17
118
-
 cefazolin pH 7.0, 22°C, mutant enzyme G240H plasmid pQE32Chl17
1160
-
 benzylpenicillin pH 7.0, 22°C, wild-type enzyme plasmid pQE32Chl17
2000
-
 benzylpenicillin pH 7.0, 22°C, mutant enzyme G240H plasmid pQE32Chl17

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7
-
 assay at plasmid pQE32Chl17

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
0.88
-
 cefotaxime pH 7.0, 22°C, wild-type enzyme plasmid pQE32Chl17
4.4
-
 cefotaxime pH 7.0, 22°C, mutant enzyme G240H plasmid pQE32Chl17
428
-
 cephalothin pH 7.0, 22°C, wild-type enzyme plasmid pQE32Chl17
531
-
 cefazolin pH 7.0, 22°C, wild-type enzyme plasmid pQE32Chl17
1242
-
 cefazolin pH 7.0, 22°C, mutant enzyme G240H plasmid pQE32Chl17
2739
-
 cephalothin pH 7.0, 22°C, mutant enzyme G240H plasmid pQE32Chl17
4762
-
 benzylpenicillin pH 7.0, 22°C, mutant enzyme G240H plasmid pQE32Chl17
18710
-
 benzylpenicillin pH 7.0, 22°C, wild-type enzyme plasmid pQE32Chl17