Cloned (Comment) | Organism |
---|---|
gene blaC, DNA and amino acid sequence determination and analysis, recombinant expression of His6-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) | Mycobacterium tuberculosis |
Crystallization (Comment) | Organism |
---|---|
purified recombinant detagged wild-type and mutant enzymes in complex with inhibitor tebipenem, hanging drop vapor diffusion, mixing of 12 mg/ml protein with 0.1 M HEPES and 2 M NH4H2PO4, pH 4.1, in a 1:1 ratio, 10°C, microseeding for the mutant enzyme, X-ray diffraction structure determination and analysis at 1.90 A and 1.75 A resolution | Mycobacterium tuberculosis |
Protein Variants | Comment | Organism |
---|---|---|
K73A | site-directed mutagenesis, inactive mutant, substrate/inhibitor binding structure | Mycobacterium tuberculosis |
Inhibitors | Comment | Organism | Structure |
---|---|---|---|
additional information | carbapenems such as meropenem, doripenem, and ertapenem react with the enzyme to form enzyme-drug covalent complexes that are hydrolyzed extremely slowly | Mycobacterium tuberculosis | |
tebi-pivoxil | binding structure analysis, overview | Mycobacterium tuberculosis | |
tebipenem | Michaelis-Menten complex, exhibits slow tight-binding inhibition at low micromolar concentrations versus the chromogenic substrate nitrocefin, binding structure analysis, overview. TBPM-PI is a prodrug that is quickly hydrolyzed to tebipenem (TEBI). Active site of BlaC-tebipenem Michaelis-Menten complex showing interactions and interatomic distances between tebipenem and active site residues, overview | Mycobacterium tuberculosis |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.0002 | - |
ertapenem | pH 6.5, 22°C, recombinant enzyme | Mycobacterium tuberculosis | |
0.0002 | - |
doripenem | pH 6.5, 22°C, recombinant enzyme | Mycobacterium tuberculosis | |
0.0008 | - |
tebipenem | pH 6.5, 22°C, recombinant enzyme | Mycobacterium tuberculosis | |
0.0034 | - |
meropenem | pH 6.5, 22°C, recombinant enzyme | Mycobacterium tuberculosis |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Mycobacterium tuberculosis | P9WKD3 | gene blaC | - |
Mycobacterium tuberculosis H37Rv | P9WKD3 | gene blaC | - |
Purification (Comment) | Organism |
---|---|
recombinant His6-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, tag cleavage by thrombin, and gel filtration | Mycobacterium tuberculosis |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
doripenem + H2O | very low activity | Mycobacterium tuberculosis | (4R,5S)-5-[(1S,2R)-1-carboxy-2-hydroxypropyl]-4-methyl-3-([(3S,5S)-5-[(sulfamoylamino)methyl]pyrrolidin-3-yl]sulfanyl)-4,5-dihydro-1H-pyrrole-2-carboxylic acid | - |
? | |
doripenem + H2O | very low activity | Mycobacterium tuberculosis H37Rv | (4R,5S)-5-[(1S,2R)-1-carboxy-2-hydroxypropyl]-4-methyl-3-([(3S,5S)-5-[(sulfamoylamino)methyl]pyrrolidin-3-yl]sulfanyl)-4,5-dihydro-1H-pyrrole-2-carboxylic acid | - |
? | |
ertapenem + H2O | very low activity | Mycobacterium tuberculosis | (4R,5S)-5-[(1S,2R)-1-carboxy-2-hydroxypropyl]-3-([(3S,5S)-5-[(3-carboxyphenyl)carbamoyl]pyrrolidin-3-yl]sulfanyl)-4-methyl-4,5-dihydro-1H-pyrrole-2-carboxylic acid | - |
? | |
ertapenem + H2O | very low activity | Mycobacterium tuberculosis H37Rv | (4R,5S)-5-[(1S,2R)-1-carboxy-2-hydroxypropyl]-3-([(3S,5S)-5-[(3-carboxyphenyl)carbamoyl]pyrrolidin-3-yl]sulfanyl)-4-methyl-4,5-dihydro-1H-pyrrole-2-carboxylic acid | - |
? | |
meropenem + H2O | very low activity | Mycobacterium tuberculosis | (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 | - |
? | |
meropenem + H2O | very low activity | Mycobacterium tuberculosis H37Rv | (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 | enzyme BlaC catalyzes the opening of the beta-lactam ring via nucleophilic attack by an active site serine residue (Ser70) to generate the acyl enzyme followed by hydrolysis of the ester bond to generate the inactive ring-opened product. Enzyme BlaC has an extremely broad spectrum of activity against penicillins and cephalosporins but weak activity against newer carbapenems. Carbapenems such as meropenem, doripenem, and ertapenem react with the enzyme to form enzyme-drug covalent complexes that are hydrolyzed extremely slowly | Mycobacterium tuberculosis | ? | - |
? | |
additional information | enzyme BlaC catalyzes the opening of the beta-lactam ring via nucleophilic attack by an active site serine residue (Ser70) to generate the acyl enzyme followed by hydrolysis of the ester bond to generate the inactive ring-opened product. Enzyme BlaC has an extremely broad spectrum of activity against penicillins and cephalosporins but weak activity against newer carbapenems. Carbapenems such as meropenem, doripenem, and ertapenem react with the enzyme to form enzyme-drug covalent complexes that are hydrolyzed extremely slowly | Mycobacterium tuberculosis H37Rv | ? | - |
? | |
nitrocefin + H2O | - |
Mycobacterium tuberculosis | (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 | - |
? | |
nitrocefin + H2O | - |
Mycobacterium tuberculosis H37Rv | (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 | - |
? | |
tebipenem + H2O | very low activity, active site of BlaC-tebipenem Michaelis-Menten complex showing interactions and interatomic distances between tebipenem and active site residues, overview | Mycobacterium tuberculosis | (4R,5S)-5-[(1S,2R)-1-carboxy-2-hydroxypropyl]-3-[[1-(4,5-dihydro-1,3-thiazol-2-yl)azetidin-3-yl]sulfanyl]-4-methyl-4,5-dihydro-1H-pyrrole-2-carboxylic acid | - |
? |
Synonyms | Comment | Organism |
---|---|---|
BlaC | - |
Mycobacterium tuberculosis |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
22 | - |
assay at room temperature | Mycobacterium tuberculosis |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.00033 | - |
ertapenem | pH 6.5, 22°C, recombinant enzyme | Mycobacterium tuberculosis | |
0.00033 | - |
doripenem | pH 6.5, 22°C, recombinant enzyme | Mycobacterium tuberculosis | |
0.0005 | - |
tebipenem | pH 6.5, 22°C, recombinant enzyme | Mycobacterium tuberculosis | |
0.0013 | - |
meropenem | pH 6.5, 22°C, recombinant enzyme | Mycobacterium tuberculosis |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
6.5 | - |
assay at | Mycobacterium tuberculosis |
General Information | Comment | Organism |
---|---|---|
evolution | BlaC is a class A beta-lactamase | Mycobacterium tuberculosis |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.3 | - |
meropenem | pH 6.5, 22°C, recombinant enzyme | Mycobacterium tuberculosis | |
0.63 | - |
tebipenem | pH 6.5, 22°C, recombinant enzyme | Mycobacterium tuberculosis | |
1.67 | - |
ertapenem | pH 6.5, 22°C, recombinant enzyme | Mycobacterium tuberculosis | |
1.67 | - |
doripenem | pH 6.5, 22°C, recombinant enzyme | Mycobacterium tuberculosis |