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Literature summary for 1.14.20.1 extracted from

  • Lee, H.J.; Dai, Y.F.; Shiau, C.Y.; Schofield, C.J.; Lloyd, M.D.
    The kinetic properties of various R258 mutants of deacetoxycephalosporin C synthase (2003), Eur. J. Biochem., 270, 1301-1307.
    View publication on PubMed

Cloned(Commentary)

Cloned (Comment) Organism
expression of wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) Streptomyces clavuligerus

Protein Variants

Protein Variants Comment Organism
R258A wild-type enzyme has a requirement for 2-oxoglutarate and cannot efficiently use hydrophobic 2-oxoacids, the mutant enzyme has broadened cosubstrate selectivity and is able to utilize hydrophobic 2-oxoacids. The efficiency of 2-oxoglutarate utilization is decreased as compared to the wild-type enzyme Streptomyces clavuligerus
R258A site-directed mutagenesis, broadened specificity for the cosubstrate compared to the wild-type enzyme, mutant enzyme can utilize hydrophobic 2-oxoacids, activity is decreased compared to the wild-type enzyme Streptomyces clavuligerus
R258F wild-type enzyme has a requirement for 2-oxoglutarate and cannot efficiently use hydrophobic 2-oxoacids, the mutant enzyme has broadened cosubstrate selectivity and is able to utilize hydrophobic 2-oxoacids. The efficiency of 2-oxoglutarate utilization is decreased as compared to the wild-type enzyme Streptomyces clavuligerus
R258F site-directed mutagenesis, broadened specificity for the cosubstrate compared to the wild-type enzyme, mutant enzyme can utilize hydrophobic 2-oxoacids, activity is decreased compared to the wild-type enzyme Streptomyces clavuligerus
R258H wild-type enzyme has a requirement for 2-oxoglutarate and cannot efficiently use hydrophobic 2-oxoacids, the mutant enzyme has broadened cosubstrate selectivity and is able to utilize hydrophobic 2-oxoacids. The efficiency of 2-oxoglutarate utilization is decreased as compared to the wild-type enzyme Streptomyces clavuligerus
R258H site-directed mutagenesis, broadened specificity for the cosubstrate compared to the wild-type enzyme, mutant enzyme can utilize hydrophobic 2-oxoacids, activity is decreased compared to the wild-type enzyme Streptomyces clavuligerus
R258K site-directed mutagenesis, broadened specificity for the cosubstrate compared to the wild-type enzyme, mutant enzyme can utilize hydrophobic 2-oxoacids, activity is decreased compared to the wild-type enzyme Streptomyces clavuligerus
R258L wild-type enzyme has a requirement for 2-oxoglutarate and cannot efficiently use hydrophobic 2-oxoacids, the mutant enzyme has broadened cosubstrate selectivity and is able to utilize hydrophobic 2-oxoacids. The efficiency of 2-oxoglutarate utilization is decreased as compared to the wild-type enzyme Streptomyces clavuligerus
R258L site-directed mutagenesis, broadened specificity for the cosubstrate compared to the wild-type enzyme, mutant enzyme can utilize hydrophobic 2-oxoacids, activity is decreased compared to the wild-type enzyme Streptomyces clavuligerus
R258Q site-directed mutagenesis, broadened specificity for the cosubstrate compared to the wild-type enzyme, mutant enzyme can utilize hydrophobic 2-oxoacids, activity is decreased compared to the wild-type enzyme Streptomyces clavuligerus

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
additional information KM-values for mutant enzymes with penicillin G or Ampicillin as prime substrate and 2-oxo-4-methylpentanoate, 2-oxoglutarate or 2-oxohexanoate as cosubstrate Streptomyces clavuligerus
0.79
-
ampicillin pH 7.5, cosubstrate 2-oxo-4-methyl-pentanoate, recombinant mutant R258F Streptomyces clavuligerus
0.79
-
penicillin G pH 7.5, cosubstrate 2-oxohexanoate, recombinant mutants R258A and R258Q Streptomyces clavuligerus
0.84
-
penicillin G pH 7.5, cosubstrate 2-oxo-4-methyl-pentanoate, recombinant mutant R258F Streptomyces clavuligerus
1.1
-
penicillin G wild-type enzyme Streptomyces clavuligerus
1.1
-
penicillin G pH 7.5, cosubstrate 2-oxoglutarate, recombinant wild-type enzyme Streptomyces clavuligerus
1.2
-
penicillin G pH 7.5, cosubstrate 2-oxo-4-methyl-pentanoate, recombinant mutant R258L Streptomyces clavuligerus
1.3
-
ampicillin pH 7.5, cosubstrate 2-oxo-4-methyl-pentanoate, recombinant mutant R258L Streptomyces clavuligerus
1.5
-
penicillin G pH 7.5, cosubstrate 2-oxo-4-methyl-pentanoate, recombinant mutants R258H and R258Q Streptomyces clavuligerus
1.6
-
penicillin G pH 7.5, cosubstrate 2-oxohexanoate, recombinant mutant R258F Streptomyces clavuligerus
1.7
-
penicillin G pH 7.5, cosubstrate 2-oxohexanoate, recombinant mutant R258L Streptomyces clavuligerus
1.8
-
penicillin G pH 7.5, cosubstrate 2-oxo-4-methyl-pentanoate, recombinant mutant R258A Streptomyces clavuligerus
1.9
-
ampicillin pH 7.5, cosubstrate 2-oxohexanoate, recombinant mutant R258F Streptomyces clavuligerus
2.6
-
ampicillin wild-type enzyme Streptomyces clavuligerus
2.6
-
ampicillin pH 7.5, cosubstrate 2-oxoglutarate, recombinant wild-type enzyme Streptomyces clavuligerus
2.6
-
ampicillin pH 7.5, cosubstrate 2-oxohexanoate, recombinant mutant R258L Streptomyces clavuligerus
3
-
penicillin G pH 7.5, cosubstrate 2-oxohexanoate, recombinant mutants R258H Streptomyces clavuligerus
3.3
-
ampicillin pH 7.5, cosubstrate 2-oxo-4-methyl-pentanoate, recombinant mutant R258Q Streptomyces clavuligerus
4.1
-
ampicillin pH 7.5, cosubstrate 2-oxo-4-methyl-pentanoate, recombinant mutant R258H Streptomyces clavuligerus
4.2
-
ampicillin pH 7.5, cosubstrate 2-oxohexanoate, recombinant mutants R258H and R258A Streptomyces clavuligerus
4.5
-
ampicillin pH 7.5, cosubstrate 2-oxo-4-methyl-pentanoate, recombinant mutant R258A Streptomyces clavuligerus
4.7
-
penicillin G pH 7.5, cosubstrate 2-oxohexanoate, recombinant mutant R258K Streptomyces clavuligerus
6.4
-
penicillin G pH 7.5, cosubstrate 2-oxoglutarate, recombinant mutant R258K Streptomyces clavuligerus
6.6
-
ampicillin pH 7.5, cosubstrate 2-oxohexanoate, recombinant mutant R258Q Streptomyces clavuligerus
7
-
penicillin G pH 7.5, cosubstrate 2-oxo-4-methyl-pentanoate, recombinant mutant R258K Streptomyces clavuligerus
13
-
ampicillin pH 7.5, cosubstrate 2-oxoglutarate, recombinant mutant R258K Streptomyces clavuligerus
15
-
ampicillin pH 7.5, cosubstrate 2-oxo-4-methyl-pentanoate, recombinant mutant R258K Streptomyces clavuligerus
24
-
ampicillin pH 7.5, cosubstrate 2-oxohexanoate, recombinant mutant R258K Streptomyces clavuligerus

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
34500
-
x * 34500, about, recombinant wild-type and mutant enzymes, SDS-PAGE Streptomyces clavuligerus

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
penicillin N + 2-oxoglutarate + O2 Streptomyces clavuligerus key step in the cephamycin C biosynthesis pathway, the ring expansion step by incorporation of a methyl group into the cephem ring deacetoxycephalosporin C + succinate + CO2 + H2O
-
ir

Organism

Organism UniProt Comment Textmining
Streptomyces clavuligerus
-
recombinant enzyme expressed in Escherichia coli
-
Streptomyces clavuligerus P18548
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by anion exchange chromatography and gel filtration Streptomyces clavuligerus
recombinant wild-type enzyme and mutant enzymes R258K, R258H, R258A, R258L and R258F Streptomyces clavuligerus

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
additional information
-
activity with different substrates and assay methods of recombinant wild-type and mutant enzymes, overview Streptomyces clavuligerus
190
-
purified recombinant wild-type Streptomyces clavuligerus

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
ampicillin + 2-oxo-4-methyl-pentanoic acid + O2 mutants R258H, R258K, R258Q, R258A, R258L, R258F, no activity with the wild-type enzyme Streptomyces clavuligerus ?
-
ir
ampicillin + 2-oxo-4-methylpentanoate + O2 wild-type enzyme has a requirement for 2-oxoglutarate and cannot efficiently use hydrophobic 2-oxoacids, the mutants R258A, R258L, R258H and R258F have broadened cosubstrate selectivity and are able to utilize hydrophobic 2-oxoacids Streptomyces clavuligerus cephalexin + succinate + CO2 + H2O
-
?
ampicillin + 2-oxoglutarate + O2
-
Streptomyces clavuligerus cephalexin + succinate + CO2 + H2O
-
?
ampicillin + 2-oxoglutarate + O2
-
Streptomyces clavuligerus ?
-
ir
ampicillin + 2-oxohexanoate + O2 wild-type enzyme has a requirement for 2-oxoglutarate and cannot efficiently use hydrophobic 2-oxoacids, the mutants R258A, R258L, R258H and R258F have broadened cosubstrate selectivity and are able to utilize hydrophobic 2-oxoacids Streptomyces clavuligerus cephalexin + ?
-
?
ampicillin + 2-oxohexanoic acid + O2 mutants R258H, R258K, R258Q, R258A, R258L, R258F, no activity with the wild-type enzyme Streptomyces clavuligerus ?
-
ir
penicillin G + 2-oxo-4-methyl-pentanoic acid + O2 mutants R258H, R258K, R258Q, R258A, R258L, R258F, no activity with the wild-type enzyme Streptomyces clavuligerus phenylacetyl-7-aminodeacetoxycephalosporanic acid + 3-methylbutanoate + CO2 + H2O
-
ir
penicillin G + 2-oxo-4-methylpentanoate + O2 wild-type enzyme has a requirement for 2-oxoglutarate and cannot efficiently use hydrophobic 2-oxoacids, the mutants R258A, R258L, R258H and R258F have broadened cosubstrate selectivity and are able to utilize hydrophobic 2-oxoacids Streptomyces clavuligerus phenylacetyl-7-aminodeacetoxy cephalosporanic acid + ?
-
?
penicillin G + 2-oxoglutarate + O2
-
Streptomyces clavuligerus phenylacetyl-7-aminodeacetoxy cephalosporanic acid + succinate + CO2 + H2O
-
?
penicillin G + 2-oxoglutarate + O2
-
Streptomyces clavuligerus phenylacetyl-7-aminodeacetoxycephalosporanic acid + succinate + CO2 + H2O
-
ir
penicillin G + 2-oxohexanoate + O2 wild-type enzyme has a requirement for 2-oxoglutarate and cannot efficiently use hydrophobic 2-oxoacids, the mutants R258A, R258L, R258H and R258F have broadened cosubstrate selectivity and are able to utilize hydrophobic 2-oxoacids Streptomyces clavuligerus phenylacetyl-7-aminodeacetoxy cephalosporanic acid + ?
-
?
penicillin G + 2-oxohexanoic acid + O2 mutants R258H, R258K, R258Q, R258A, R258L, R258F, no activity with the wild-type enzyme Streptomyces clavuligerus phenylacetyl-7-aminodeacetoxycephalosporanic acid + pentanoate + CO2 + H2O
-
ir
penicillin N + 2-oxoglutarate + O2 wild-type enzyme has a requirement for 2-oxoglutarate and cannot efficiently use hydrophobic 2-oxoacids, the mutants R258A, R258L, R258H and R258F have broadened cosubstrate selectivity and are able to utilize hydrophobic 2-oxoacids Streptomyces clavuligerus deacetoxycephalosporin C + succinate + CO2 + H2O
-
?
penicillin N + 2-oxoglutarate + O2 key step in the cephamycin C biosynthesis pathway, the ring expansion step by incorporation of a methyl group into the cephem ring Streptomyces clavuligerus deacetoxycephalosporin C + succinate + CO2 + H2O
-
ir
penicillin N + 2-oxoglutarate + O2 specific for 2-oxoglutarate, which cannot be substituted by other 2-oxoacids e.g. 2-oxohexanoic acid, or 2-oxo-4-methyl-pentanoic acid Streptomyces clavuligerus deacetoxycephalosporin C + succinate + CO2 + H2O
-
ir

Subunits

Subunits Comment Organism
? x * 34500, about, recombinant wild-type and mutant enzymes, SDS-PAGE Streptomyces clavuligerus

Synonyms

Synonyms Comment Organism
DAOCS
-
Streptomyces clavuligerus
deacetoxycephalosporin C synthase
-
Streptomyces clavuligerus

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.001
-
penicillin G pH 7.5, cosubstrate 2-oxohexanoate, recombinant mutant R258K Streptomyces clavuligerus
0.005
-
ampicillin pH 7.5, cosubstrate 2-oxo-4-methyl-pentanoate, recombinant mutant R258H Streptomyces clavuligerus
0.007
-
ampicillin pH 7.5, cosubstrate 2-oxohexanoate, recombinant mutant R258H Streptomyces clavuligerus
0.008
-
ampicillin pH 7.5, cosubstrate 2-oxohexanoate, recombinant mutant R258A Streptomyces clavuligerus
0.009
-
ampicillin pH 7.5, cosubstrate 2-oxo-4-methyl-pentanoate, recombinant mutant R258L Streptomyces clavuligerus
0.009
-
ampicillin pH 7.5, cosubstrate 2-oxohexanoate, recombinant mutant R258K Streptomyces clavuligerus
0.01
-
penicillin G pH 7.5, cosubstrate 2-oxoglutarate, recombinant mutant R258K Streptomyces clavuligerus
0.01
-
penicillin G pH 7.5, cosubstrate 2-oxohexanoate, recombinant mutant R258Q Streptomyces clavuligerus
0.011
-
ampicillin pH 7.5, cosubstrate 2-oxo-4-methyl-pentanoate, recombinant mutants R258A and R258F Streptomyces clavuligerus
0.011
-
ampicillin pH 7.5, cosubstrate 2-oxoglutarate, recombinant mutant R258K Streptomyces clavuligerus
0.012
-
ampicillin pH 7.5, cosubstrate 2-oxohexanoate, recombinant mutants R258L and R258F Streptomyces clavuligerus
0.013
-
ampicillin pH 7.5, cosubstrate 2-oxo-4-methyl-pentanoate, recombinant mutant R258K Streptomyces clavuligerus
0.013
-
penicillin G pH 7.5, cosubstrate 2-oxohexanoate, recombinant mutants R258A and R258H Streptomyces clavuligerus
0.014
-
ampicillin wild-type enzyme Streptomyces clavuligerus
0.014
-
penicillin G pH 7.5, cosubstrate 2-oxo-4-methyl-pentanoate, recombinant mutant R258H Streptomyces clavuligerus
0.014
-
ampicillin pH 7.5, cosubstrate 2-oxo-4-methyl-pentanoate, recombinant mutant R258Q Streptomyces clavuligerus
0.014
-
ampicillin pH 7.5, cosubstrate 2-oxoglutarate, recombinant wild-type enzyme Streptomyces clavuligerus
0.017
-
ampicillin pH 7.5, cosubstrate 2-oxohexanoate, recombinant mutant R258Q Streptomyces clavuligerus
0.019
-
penicillin G pH 7.5, cosubstrate 2-oxohexanoate, recombinant mutant R258L Streptomyces clavuligerus
0.02
-
penicillin G pH 7.5, cosubstrate 2-oxo-4-methyl-pentanoate, recombinant mutants R258K, R258A, and R258L Streptomyces clavuligerus
0.021
-
penicillin G wild-type enzyme Streptomyces clavuligerus
0.021
-
penicillin G pH 7.5, cosubstrate 2-oxo-4-methyl-pentanoate, recombinant mutant R258Q Streptomyces clavuligerus
0.021
-
penicillin G pH 7.5, cosubstrate 2-oxoglutarate, recombinant wild-type enzyme Streptomyces clavuligerus
0.024
-
penicillin G pH 7.5, cosubstrate 2-oxohexanoate, recombinant mutant R258F Streptomyces clavuligerus
0.025
-
penicillin G pH 7.5, cosubstrate 2-oxo-4-methyl-pentanoate, recombinant mutant R258F Streptomyces clavuligerus

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.5
-
assay at Streptomyces clavuligerus