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Literature summary for 3.5.2.6 extracted from

  • Bansal, A.; Kar, D.; Pandey, S.D.; Matcha, A.; Kumar, N.G.; Nathan, S.; Ghosh, A.S.
    A tyrosine residue along with a glutamic acid of the omega-like loop governs the beta-lactamase activity of MSMEG_4455 in Mycobacterium smegmatis (2017), Protein J., 36, 220-227 .
    View publication on PubMed

Cloned(Commentary)

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

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

Inhibitors Comment Organism Structure
clavulanic acid
-
Mycolicibacterium smegmatis

KM Value [mM]

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/ Products (Substrates)

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

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 (Commentary)

Purification (Comment) Organism
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography Mycolicibacterium smegmatis

Substrates and Products (Substrate)

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

Subunits Comment Organism
? x * 43000, about, recombinant His-tagged wild-type and mutant enzymes, SDS-PAGE Mycolicibacterium smegmatis

Synonyms

Synonyms Comment Organism
BlaA
-
Mycolicibacterium smegmatis
MSMEG_4455
-
Mycolicibacterium smegmatis

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
30 37 assay at Mycolicibacterium smegmatis

Turnover Number [1/s]

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

pH Optimum Minimum pH Optimum Maximum Comment Organism
8
-
assay at Mycolicibacterium smegmatis

General Information

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 [mM/s]

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