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Literature summary extracted from

  • Balakrishnan, N.; Ganesan, S.; Rajasekaran, P.; Rajendran, L.; Teddu, S.; Durairaaj, M.
    Modified deacetylcephalosporin C synthase for the biotransformation of semisynthetic cephalosporins (2016), Appl. Environ. Microbiol., 82, 3711-3720 .
    View publication on PubMedView publication on EuropePMC

Activating Compound

EC Number Activating Compound Comment Organism Structure
1.14.11.26 ascorbate required Streptomyces clavuligerus

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.14.11.26 geen cefF, recombinant exression of cefF library mutants, site-directed mutants, and wild-type enzyme in Escherichia coli strain BL21(DE3) Streptomyces clavuligerus

Protein Variants

EC Number Protein Variants Comment Organism
1.14.11.26 A177V random mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme Streptomyces clavuligerus
1.14.11.26 A311V random mutagenesis Streptomyces clavuligerus
1.14.11.26 E16G/T90A/T304A random mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme Streptomyces clavuligerus
1.14.11.26 E16G/T90A/T304A site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme Streptomyces clavuligerus
1.14.11.26 E209Q random mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme Streptomyces clavuligerus
1.14.11.26 E82D random mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme Streptomyces clavuligerus
1.14.11.26 F267L random mutagenesis, active site mutation, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme Streptomyces clavuligerus
1.14.11.26 I193V random mutagenesis, active site mutation, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme Streptomyces clavuligerus
1.14.11.26 L236V random mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme Streptomyces clavuligerus
1.14.11.26 M184I random mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme Streptomyces clavuligerus
1.14.11.26 M229V random mutagenesis Streptomyces clavuligerus
1.14.11.26 additional information mutagenic cefF library creation by random mutagenesis and screening of the mutant deacetylcephalosporin C synthase enzyme library, homology modeling of DACS structure and mapping of mutant positions. Process-level biotransformation reaction of cephalosporin G to deacetylcephalosporin G by mutants of deacetylcephalosporin C synthase. Deacetylcephalosporin G can be converted completely into hydroxymethyl-7-amino-cephalosporanic acid (HACA) in about 30 min by a subsequent reaction, thus facilitating scalability toward commercialization. Directed-evolution strategies such as random, semirational, rational, and computational methods are used for systematic engineering of DACS for improved activity with cephalosporin G Streptomyces clavuligerus
1.14.11.26 P186L random mutagenesis and site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme Streptomyces clavuligerus
1.14.11.26 P72L random mutagenesis and site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme Streptomyces clavuligerus
1.14.11.26 R182S random mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme Streptomyces clavuligerus
1.14.11.26 R182W random mutagenesis, active site mutation, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme Streptomyces clavuligerus
1.14.11.26 S251F random mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme Streptomyces clavuligerus
1.14.11.26 S260G random mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme Streptomyces clavuligerus
1.14.11.26 T273A random mutagenesis Streptomyces clavuligerus
1.14.11.26 T90A random mutagenesis Streptomyces clavuligerus
1.14.11.26 T90A/A311V site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme Streptomyces clavuligerus
1.14.11.26 T90A/P72L site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme Streptomyces clavuligerus
1.14.11.26 T90A/P72L/A311V site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme Streptomyces clavuligerus
1.14.11.26 T90A/P72L/A311V/A40V/M229I/T273A site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme Streptomyces clavuligerus
1.14.11.26 T90A/P72L/A311V/A40V/M229I/T273A/M184I/I193V/F267L site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme Streptomyces clavuligerus
1.14.11.26 T90A/P72L/A311V/A40V/M229I/T273A/R182S site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme Streptomyces clavuligerus
1.14.11.26 T90A/P72L/A311V/A40V/M229I/T273A/S251F site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme Streptomyces clavuligerus
1.14.11.26 T90A/P72L/A311V/A40V/M229I/T273A/S260G site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme Streptomyces clavuligerus
1.14.11.26 T90A/P72L/A311V/A40V/M229I/T273A/V171L/F267L site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme Streptomyces clavuligerus
1.14.11.26 T90A/P72L/A311V/A40V/M229I/T273A/V171L/R182W/F267L site-directed mutagenesis, the mutant shows highly increased activity with cephalosporin G compared to the wild-type enzyme Streptomyces clavuligerus
1.14.11.26 T90A/P72L/A311V/A40V/M229I/T273A/V171L/R182W/F267L/A241V/V307A site-directed mutagenesis, the mutant shows highly increased activity with cephalosporin G compared to the wild-type enzyme Streptomyces clavuligerus
1.14.11.26 T90A/P72L/A311V/A40V/M229I/T273A/V171L/R182W/F267L/G108D site-directed mutagenesis, the mutant shows highly increased activity with cephalosporin G compared to the wild-type enzyme Streptomyces clavuligerus
1.14.11.26 T90A/P72L/A311V/A40V/M229I/T273A/V171L/R182W/F267L/N313D site-directed mutagenesis, the mutant shows highly increased activity with cephalosporin G compared to the wild-type enzyme Streptomyces clavuligerus
1.14.11.26 T90A/P72L/A311V/A40V/M229I/T273A/V171L/R182W/F267L/R91G site-directed mutagenesis, the mutant shows highly increased activity with cephalosporin G compared to the wild-type enzyme Streptomyces clavuligerus
1.14.11.26 T90A/P72L/A311V/A40V/M229I/T273A/V171L/R182W/F267L/R91G/A241V/V307A site-directed mutagenesis, the mutant shows highly increased activity with cephalosporin G compared to the wild-type enzyme Streptomyces clavuligerus
1.14.11.26 T90A/P72L/A311V/A40V/M229I/T273A/V171L/R182W/F267L/T96S/A241V/V307A site-directed mutagenesis, the mutant shows highly increased activity with cephalosporin G compared to the wild-type enzyme Streptomyces clavuligerus
1.14.11.26 T90A/P72L/A311V/A40V/M229I/T273A/V171L/R182W/F267L/T96S/G255D/A280S site-directed mutagenesis, the mutant shows highly increased activity with cephalosporin G compared to the wild-type enzyme Streptomyces clavuligerus
1.14.11.26 T90A/P72L/A311V/A40V/M229I/T273A/V171L/R182W/F267L/V226I site-directed mutagenesis, the mutant shows highly increased activity with cephalosporin G compared to the wild-type enzyme Streptomyces clavuligerus
1.14.11.26 T90A/P72L/A311V/A40V/M229I/T273A/V221P site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme Streptomyces clavuligerus
1.14.11.26 T90A/P72L/A311V/F195L site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme Streptomyces clavuligerus
1.14.11.26 T90A/P72L/A311V/P7L/A237V site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme Streptomyces clavuligerus
1.14.11.26 T90A/P72L/A311V/P7L/T273A site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme Streptomyces clavuligerus
1.14.11.26 T90A/P72L/A311V/R250L site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme Streptomyces clavuligerus
1.14.11.26 T90A/P72L/A311V/V206I/A210V site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme Streptomyces clavuligerus
1.14.11.26 T90A/P72L/A311V/V206I/A210V/T273A site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme Streptomyces clavuligerus
1.14.11.26 V150A random mutagenesis Streptomyces clavuligerus
1.14.11.26 V171L random mutagenesis, active site mutation, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme Streptomyces clavuligerus
1.14.11.26 V171M random mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme Streptomyces clavuligerus
1.14.11.26 V221A random mutagenesis Streptomyces clavuligerus
1.14.11.26 V221H site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme Streptomyces clavuligerus
1.14.11.26 V221P site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme Streptomyces clavuligerus
1.14.11.26 V221T random mutagenesis and site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme Streptomyces clavuligerus
1.14.11.26 V249I random mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme Streptomyces clavuligerus
1.14.11.26 Y38C random mutagenesis Streptomyces clavuligerus

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.14.11.26 2.58
-
cephalosporin G pH 7.5, 25°C, recombinant wild-type enzyme Streptomyces clavuligerus
1.14.11.26 2.58
-
cephalosporin G pH 7.5, 25°C, recombinant mutant T90A/P72L/A311V Streptomyces clavuligerus
1.14.11.26 4.41
-
cephalosporin G pH 7.5, 25°C, recombinant mutant T90A/P72L/A311V/A40V/M229I/T273A/V171L/R182W/F267L/G108D Streptomyces clavuligerus
1.14.11.26 4.64
-
cephalosporin G pH 7.5, 25°C, recombinant mutant T90A/P72L/A311V/A40V/M229I/T273A Streptomyces clavuligerus
1.14.11.26 7.24
-
cephalosporin G pH 7.5, 25°C, recombinant mutant T90A/P72L/A311V/A40V/M229I/T273A/V171L/R182W/F267L Streptomyces clavuligerus
1.14.11.26 9.2
-
cephalosporin G pH 7.5, 25°C, recombinant mutant T90A/P72L/A311V/A40V/M229I/T273A/V171L/R182W/F267L Streptomyces clavuligerus

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
1.14.11.26 Fe2+ dependent on, required for catalysis Streptomyces clavuligerus

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.14.11.26 deacetoxycephalosporin C + 2-oxoglutarate + O2 Streptomyces clavuligerus
-
deacetylcephalosporin C + succinate + CO2
-
?
1.14.11.26 deacetoxycephalosporin C + 2-oxoglutarate + O2 Streptomyces clavuligerus ATCC 27064
-
deacetylcephalosporin C + succinate + CO2
-
?

Organism

EC Number Organism UniProt Comment Textmining
1.14.11.26 Streptomyces clavuligerus P42220
-
-
1.14.11.26 Streptomyces clavuligerus ATCC 27064 P42220
-
-

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.14.11.26 cephalosporin G + 2-oxoglutarate + O2 the wild-type enzyme shows negligible activity toward cephalosporin G, an unnatural and less expensive substrate analogue, but diverse enzyme mutants show increased to high activity with cephalosporin G, overview Streptomyces clavuligerus deacetylcephalosporin G + succinate + CO2
-
?
1.14.11.26 cephalosporin G + 2-oxoglutarate + O2 the wild-type enzyme shows negligible activity toward cephalosporin G, an unnatural and less expensive substrate analogue, but diverse enzyme mutants show increased to high activity with cephalosporin G, overview Streptomyces clavuligerus ATCC 27064 deacetylcephalosporin G + succinate + CO2
-
?
1.14.11.26 deacetoxycephalosporin C + 2-oxoglutarate + O2
-
Streptomyces clavuligerus deacetylcephalosporin C + succinate + CO2
-
?
1.14.11.26 deacetoxycephalosporin C + 2-oxoglutarate + O2
-
Streptomyces clavuligerus ATCC 27064 deacetylcephalosporin C + succinate + CO2
-
?

Synonyms

EC Number Synonyms Comment Organism
1.14.11.26 cefF
-
Streptomyces clavuligerus
1.14.11.26 DACS
-
Streptomyces clavuligerus
1.14.11.26 deacetylcephalosporin C synthase
-
Streptomyces clavuligerus

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.14.11.26 25
-
assay at Streptomyces clavuligerus

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.14.11.26 0.055
-
cephalosporin G pH 7.5, 25°C, recombinant wild-type enzyme Streptomyces clavuligerus
1.14.11.26 0.122
-
cephalosporin G pH 7.5, 25°C, recombinant mutant T90A/P72L/A311V Streptomyces clavuligerus
1.14.11.26 0.326
-
cephalosporin G pH 7.5, 25°C, recombinant mutant T90A/P72L/A311V/A40V/M229I/T273A Streptomyces clavuligerus
1.14.11.26 1.805
-
cephalosporin G pH 7.5, 25°C, recombinant mutant T90A/P72L/A311V/A40V/M229I/T273A/V171L/R182W/F267L Streptomyces clavuligerus
1.14.11.26 1.899
-
cephalosporin G pH 7.5, 25°C, recombinant mutant T90A/P72L/A311V/A40V/M229I/T273A/V171L/R182W/F267L/G108D Streptomyces clavuligerus
1.14.11.26 2.302
-
cephalosporin G pH 7.5, 25°C, recombinant mutant T90A/P72L/A311V/A40V/M229I/T273A/V171L/R182W/F267L Streptomyces clavuligerus

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.14.11.26 7.5
-
assay at Streptomyces clavuligerus

General Information

EC Number General Information Comment Organism
1.14.11.26 evolution the enzyme belongs to the family of 2-oxoglutarate-dependent oxygenases Streptomyces clavuligerus
1.14.11.26 metabolism deacetylcephalosporin C synthase (DACS) transforms an inert methyl group of deacetoxycephalosporin C (DAOC) into an active hydroxyl group of deacetylcephalosporin C (DAC) during the biosynthesis of cephalosporins Streptomyces clavuligerus
1.14.11.26 additional information homology modeling of DACS structure Streptomyces clavuligerus
1.14.11.26 physiological function the enzyme is involved in the biosynthesis of cephalosporins, it carries out a critical transformation of an inert cephem methyl group of deacetoxycephalosporin C (DAOC) into an active hydroxyl group of deacetylcephalosporin C (DAC) in the presence of iron, oxygen, ascorbic acid, and 2-oxoglutarate. It is a step which is chemically difficult to accomplish and which offers a second substitution site for derivatization in cephalosporins Streptomyces clavuligerus

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
1.14.11.26 0.021
-
cephalosporin G pH 7.5, 25°C, recombinant wild-type enzyme Streptomyces clavuligerus
1.14.11.26 0.047
-
cephalosporin G pH 7.5, 25°C, recombinant mutant T90A/P72L/A311V Streptomyces clavuligerus
1.14.11.26 0.07
-
cephalosporin G pH 7.5, 25°C, recombinant mutant T90A/P72L/A311V/A40V/M229I/T273A Streptomyces clavuligerus
1.14.11.26 0.249
-
cephalosporin G pH 7.5, 25°C, recombinant mutant T90A/P72L/A311V/A40V/M229I/T273A/V171L/R182W/F267L Streptomyces clavuligerus
1.14.11.26 0.25
-
cephalosporin G pH 7.5, 25°C, recombinant mutant T90A/P72L/A311V/A40V/M229I/T273A/V171L/R182W/F267L Streptomyces clavuligerus
1.14.11.26 0.43
-
cephalosporin G pH 7.5, 25°C, recombinant mutant T90A/P72L/A311V/A40V/M229I/T273A/V171L/R182W/F267L/G108D Streptomyces clavuligerus