Cloned (Comment) | Organism |
---|---|
gene MSMEG_4455, DNA and amino acid sequence determination and analysis, recombinant ectopic expression of wild-type and mutant enzymes in beta-lactamase-deficient Escherichia coli mutant strain AM1OC-1, recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) | Mycolicibacterium smegmatis |
Protein Variants | Comment | Organism |
---|---|---|
E193A | site-directed mutagenesis, the mutant shows 30% reduced activity compared to the wild-type enzyme. In vivo expression of 4455E193A reduces the beta-lactam resistance for penicillin and ampicillin by twofold as compared to the expression of wild-type MSMEG_4455 | Mycolicibacterium smegmatis |
Y194F | site-directed mutagenesis, the mutant shows 62% reduced activity compared to the wild-type enzyme | Mycolicibacterium smegmatis |
Inhibitors | Comment | Organism | Structure |
---|---|---|---|
clavulanic acid | - |
Mycolicibacterium smegmatis |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | Michaelis-Menten kinetics | Mycolicibacterium smegmatis | |
0.00335 | - |
penicillin G | pH 8.0, 30°C, recombinant wild-type enzyme | Mycolicibacterium smegmatis | |
0.00649 | - |
amoxicillin | pH 8.0, 30°C, recombinant wild-type enzyme | Mycolicibacterium smegmatis | |
0.00792 | - |
ticarcillin | pH 8.0, 30°C, recombinant wild-type enzyme | Mycolicibacterium smegmatis | |
0.00897 | - |
cephalexin | pH 8.0, 30°C, recombinant wild-type enzyme | Mycolicibacterium smegmatis | |
0.00948 | - |
nitrocefin | pH 8.0, 30°C, recombinant wild-type enzyme | Mycolicibacterium smegmatis | |
0.00954 | - |
ampicillin | pH 8.0, 30°C, recombinant wild-type enzyme | Mycolicibacterium smegmatis | |
0.01176 | - |
ceftriaxone | pH 8.0, 30°C, recombinant wild-type enzyme | Mycolicibacterium smegmatis | |
0.01256 | - |
piperacillin | pH 8.0, 30°C, recombinant wild-type enzyme | Mycolicibacterium smegmatis | |
0.02174 | - |
carbenicillin | pH 8.0, 30°C, recombinant wild-type enzyme | Mycolicibacterium smegmatis | |
0.1931 | - |
Oxacillin | pH 8.0, 30°C, recombinant wild-type enzyme | Mycolicibacterium smegmatis | |
0.3647 | - |
cefotaxime | pH 8.0, 30°C, recombinant wild-type enzyme | Mycolicibacterium smegmatis | |
0.655 | - |
cephalothin | pH 8.0, 30°C, recombinant wild-type enzyme | Mycolicibacterium smegmatis | |
36.46 | - |
Bocillin-FL | pH 8.0, 30°C, recombinant enzyme Y194F | Mycolicibacterium smegmatis | |
58.28 | - |
Bocillin-FL | pH 8.0, 30°C, recombinant enzyme E193A | Mycolicibacterium smegmatis |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
ampicillin + H2O | Mycolicibacterium smegmatis | - |
(2R,4S)-2-[(R)-[[(2R)-2-amino-2-phenylacetyl]amino](carboxy)methyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxylic acid | - |
? | |
ampicillin + H2O | Mycolicibacterium smegmatis ATCC 700084 | - |
(2R,4S)-2-[(R)-[[(2R)-2-amino-2-phenylacetyl]amino](carboxy)methyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxylic acid | - |
? | |
ampicillin + H2O | Mycolicibacterium smegmatis mc(2)155 | - |
(2R,4S)-2-[(R)-[[(2R)-2-amino-2-phenylacetyl]amino](carboxy)methyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxylic acid | - |
? | |
penicillin G + H2O | Mycolicibacterium smegmatis | - |
(2R,4S)-2-[(R)-carboxy[(phenylacetyl)amino]methyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxylic acid | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Mycolicibacterium smegmatis | A0R0P0 | i.e. Mycobacterium smegmatis | - |
Mycolicibacterium smegmatis ATCC 700084 | A0R0P0 | i.e. Mycobacterium smegmatis | - |
Mycolicibacterium smegmatis mc(2)155 | A0R0P0 | i.e. Mycobacterium smegmatis | - |
Purification (Comment) | Organism |
---|---|
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography | Mycolicibacterium smegmatis |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
amoxicillin + H2O | - |
Mycolicibacterium smegmatis | (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 | - |
Mycolicibacterium smegmatis ATCC 700084 | (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 | - |
Mycolicibacterium smegmatis mc(2)155 | (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 | - |
Mycolicibacterium smegmatis | (2R,4S)-2-[(R)-[[(2R)-2-amino-2-phenylacetyl]amino](carboxy)methyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxylic acid | - |
? | |
ampicillin + H2O | - |
Mycolicibacterium smegmatis ATCC 700084 | (2R,4S)-2-[(R)-[[(2R)-2-amino-2-phenylacetyl]amino](carboxy)methyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxylic acid | - |
? | |
ampicillin + H2O | - |
Mycolicibacterium smegmatis mc(2)155 | (2R,4S)-2-[(R)-[[(2R)-2-amino-2-phenylacetyl]amino](carboxy)methyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxylic acid | - |
? | |
Bocillin-FL + H2O | activity analysis with Bocillin-FL, a fluorescent penicillin | Mycolicibacterium smegmatis | ? | - |
? | |
carbenicillin + H2O | - |
Mycolicibacterium smegmatis | (2R,4S)-2-((R)-carboxy[2-carboxy(phenyl)acetamido]methyl)-5,5-dimethyl-1,3-thiazolidine-4-carboxylic acid | - |
? | |
cefotaxime + H2O | - |
Mycolicibacterium smegmatis | (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 | - |
Mycolicibacterium smegmatis ATCC 700084 | (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 | - |
Mycolicibacterium smegmatis mc(2)155 | (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 | - |
? | |
ceftriaxone + H2O | - |
Mycolicibacterium smegmatis | (2R)-2-[(R)-[[(2Z)-2-(2-amino-1,3-thiazol-4-yl)-2-(methoxyimino)acetyl]amino](carboxy)methyl]-5-[[(6-hydroxy-2-methyl-5-oxo-2,5-dihydro-1,2,4-triazin-3-yl)sulfanyl]methyl]-3,6-dihydro-2H-1,3-thiazine-4-carboxylic acid | - |
? | |
cephalexin + H2O | - |
Mycolicibacterium smegmatis | (2R)-2-[(R)-[[(2R)-2-amino-2-phenylacetyl]amino](carboxy)methyl]-5-methyl-3,6-dihydro-2H-1,3-thiazine-4-carboxylic acid | - |
? | |
cephalexin + H2O | - |
Mycolicibacterium smegmatis ATCC 700084 | (2R)-2-[(R)-[[(2R)-2-amino-2-phenylacetyl]amino](carboxy)methyl]-5-methyl-3,6-dihydro-2H-1,3-thiazine-4-carboxylic acid | - |
? | |
cephalexin + H2O | - |
Mycolicibacterium smegmatis mc(2)155 | (2R)-2-[(R)-[[(2R)-2-amino-2-phenylacetyl]amino](carboxy)methyl]-5-methyl-3,6-dihydro-2H-1,3-thiazine-4-carboxylic acid | - |
? | |
cephalothin + H2O | - |
Mycolicibacterium smegmatis | (2R)-5-[(acetyloxy)methyl]-2-[(R)-carboxy[2-(thiophen-2-yl)acetamido]methyl]-3,6-dihydro-2H-1,3-thiazine-4-carboxylic acid | - |
? | |
L-Ala-gamma-D-Glu-L-Lys-D-Ala-D-Ala + H2O | peptidoglycan-mimetic pentapeptide substrate | Mycolicibacterium smegmatis | ? | - |
? | |
additional information | no activity with aztreonam and imipenem | Mycolicibacterium smegmatis | ? | - |
- |
|
additional information | no activity with aztreonam and imipenem | Mycolicibacterium smegmatis ATCC 700084 | ? | - |
- |
|
additional information | no activity with aztreonam and imipenem | Mycolicibacterium smegmatis mc(2)155 | ? | - |
- |
|
Nepsilon-diacetyl-L-Lys-D-Ala-D-Ala + H2O | artificial substrate | Mycolicibacterium smegmatis | ? | - |
? | |
nitrocefin + H2O | - |
Mycolicibacterium smegmatis | (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 | - |
? | |
oxacillin + H2O | - |
Mycolicibacterium smegmatis | (2R,4S)-2-((R)-carboxy[(5-methyl-3-phenyl-1,2-oxazole-4-carbonyl)amino]methyl)-5,5-dimethyl-1,3-thiazolidine-4-carboxylic acid | - |
? | |
penicillin G + H2O | - |
Mycolicibacterium smegmatis | (2R,4S)-2-[(R)-carboxy[(phenylacetyl)amino]methyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxylic acid | - |
? | |
piperacillin + H2O | - |
Mycolicibacterium smegmatis | (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 | - |
? | |
ticarcillin + H2O | - |
Mycolicibacterium smegmatis | (2R,4S)-2-[(R)-carboxy[[(2R)-2-carboxy-2-(thiophen-3-yl)acetyl]amino]methyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxylic acid | - |
? |
Subunits | Comment | Organism |
---|---|---|
? | x * 43000, about, recombinant His-tagged wild-type and mutant enzymes, SDS-PAGE | Mycolicibacterium smegmatis |
Synonyms | Comment | Organism |
---|---|---|
BlaA | - |
Mycolicibacterium smegmatis |
MSMEG_4455 | - |
Mycolicibacterium smegmatis |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
30 | 37 | assay at | Mycolicibacterium smegmatis |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.014 | - |
nitrocefin | pH 8.0, 30°C, recombinant wild-type enzyme | Mycolicibacterium smegmatis | |
0.079 | - |
cephalexin | pH 8.0, 30°C, recombinant wild-type enzyme | Mycolicibacterium smegmatis | |
1.031 | - |
cephalothin | pH 8.0, 30°C, recombinant wild-type enzyme | Mycolicibacterium smegmatis | |
1.572 | - |
cefotaxime | pH 8.0, 30°C, recombinant wild-type enzyme | Mycolicibacterium smegmatis | |
1.722 | - |
ticarcillin | pH 8.0, 30°C, recombinant wild-type enzyme | Mycolicibacterium smegmatis | |
1.77 | - |
amoxicillin | pH 8.0, 30°C, recombinant wild-type enzyme | Mycolicibacterium smegmatis | |
1.825 | - |
ceftriaxone | pH 8.0, 30°C, recombinant wild-type enzyme | Mycolicibacterium smegmatis | |
3.72 | - |
Bocillin-FL | pH 8.0, 30°C, recombinant enzyme Y194F | Mycolicibacterium smegmatis | |
9.62 | - |
ampicillin | pH 8.0, 30°C, recombinant wild-type enzyme | Mycolicibacterium smegmatis | |
10.22 | - |
carbenicillin | pH 8.0, 30°C, recombinant wild-type enzyme | Mycolicibacterium smegmatis | |
11.9 | - |
Oxacillin | pH 8.0, 30°C, recombinant wild-type enzyme | Mycolicibacterium smegmatis | |
12.03 | - |
Bocillin-FL | pH 8.0, 30°C, recombinant enzyme E193A | Mycolicibacterium smegmatis | |
22.02 | - |
piperacillin | pH 8.0, 30°C, recombinant wild-type enzyme | Mycolicibacterium smegmatis | |
27.6 | - |
penicillin G | pH 8.0, 30°C, recombinant wild-type enzyme | Mycolicibacterium smegmatis |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
8 | - |
assay at | Mycolicibacterium smegmatis |
General Information | Comment | Organism |
---|---|---|
evolution | the purified soluble form of recombinant MSMEG_4455 shows a beta-lactam hydrolysis pattern similar to group 2a penicillinase. The BlaA enzyme of H37Ra is identical to BlaC of H37Rv | Mycolicibacterium smegmatis |
additional information | enzyme structure-function relationship in silico analysis, overview. Residues E193 and Y194 of the omega-like loop might have importance in strengthening hydrogen bond network around the active site, though involvement of tyrosine is rare for beta-lactamase activity. Y194 is the key for beta-lactamase activity of MSMEG_4455 | Mycolicibacterium smegmatis |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.102 | - |
Bocillin-FL | pH 8.0, 30°C, recombinant enzyme Y194F | Mycolicibacterium smegmatis | |
0.206 | - |
Bocillin-FL | pH 8.0, 30°C, recombinant enzyme E193A | Mycolicibacterium smegmatis | |
1.48 | - |
nitrocefin | pH 8.0, 30°C, recombinant wild-type enzyme | Mycolicibacterium smegmatis | |
1.57 | - |
cephalothin | pH 8.0, 30°C, recombinant wild-type enzyme | Mycolicibacterium smegmatis | |
4.31 | - |
cefotaxime | pH 8.0, 30°C, recombinant wild-type enzyme | Mycolicibacterium smegmatis | |
8.81 | - |
cephalexin | pH 8.0, 30°C, recombinant wild-type enzyme | Mycolicibacterium smegmatis | |
61.6 | - |
Oxacillin | pH 8.0, 30°C, recombinant wild-type enzyme | Mycolicibacterium smegmatis | |
155.2 | - |
ceftriaxone | pH 8.0, 30°C, recombinant wild-type enzyme | Mycolicibacterium smegmatis | |
217.4 | - |
ticarcillin | pH 8.0, 30°C, recombinant wild-type enzyme | Mycolicibacterium smegmatis | |
272.7 | - |
amoxicillin | pH 8.0, 30°C, recombinant wild-type enzyme | Mycolicibacterium smegmatis | |
470.1 | - |
carbenicillin | pH 8.0, 30°C, recombinant wild-type enzyme | Mycolicibacterium smegmatis | |
1008.4 | - |
ampicillin | pH 8.0, 30°C, recombinant wild-type enzyme | Mycolicibacterium smegmatis | |
1753.2 | - |
piperacillin | pH 8.0, 30°C, recombinant wild-type enzyme | Mycolicibacterium smegmatis | |
8238.8 | - |
penicillin G | pH 8.0, 30°C, recombinant wild-type enzyme | Mycolicibacterium smegmatis |