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Literature summary for 1.14.11.26 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

Activating Compound Comment Organism Structure
ascorbate required Streptomyces clavuligerus

Cloned(Commentary)

Cloned (Comment) Organism
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

Protein Variants Comment Organism
A177V random mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme Streptomyces clavuligerus
A311V random mutagenesis Streptomyces clavuligerus
E16G/T90A/T304A random mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme Streptomyces clavuligerus
E16G/T90A/T304A site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme Streptomyces clavuligerus
E209Q random mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme Streptomyces clavuligerus
E82D random mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme Streptomyces clavuligerus
F267L random mutagenesis, active site mutation, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme Streptomyces clavuligerus
I193V random mutagenesis, active site mutation, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme Streptomyces clavuligerus
L236V random mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme Streptomyces clavuligerus
M184I random mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme Streptomyces clavuligerus
M229V random mutagenesis Streptomyces clavuligerus
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
P186L random mutagenesis and site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme Streptomyces clavuligerus
P72L random mutagenesis and site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme Streptomyces clavuligerus
R182S random mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme Streptomyces clavuligerus
R182W random mutagenesis, active site mutation, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme Streptomyces clavuligerus
S251F random mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme Streptomyces clavuligerus
S260G random mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme Streptomyces clavuligerus
T273A random mutagenesis Streptomyces clavuligerus
T90A random mutagenesis Streptomyces clavuligerus
T90A/A311V site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme Streptomyces clavuligerus
T90A/P72L site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme Streptomyces clavuligerus
T90A/P72L/A311V site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme Streptomyces clavuligerus
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
T90A/P72L/A311V/F195L site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme Streptomyces clavuligerus
T90A/P72L/A311V/P7L/A237V site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme Streptomyces clavuligerus
T90A/P72L/A311V/P7L/T273A site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme Streptomyces clavuligerus
T90A/P72L/A311V/R250L site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme Streptomyces clavuligerus
T90A/P72L/A311V/V206I/A210V site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme Streptomyces clavuligerus
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
V150A random mutagenesis Streptomyces clavuligerus
V171L random mutagenesis, active site mutation, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme Streptomyces clavuligerus
V171M random mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme Streptomyces clavuligerus
V221A random mutagenesis Streptomyces clavuligerus
V221H site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme Streptomyces clavuligerus
V221P site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme Streptomyces clavuligerus
V221T random mutagenesis and site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme Streptomyces clavuligerus
V249I random mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme Streptomyces clavuligerus
Y38C random mutagenesis Streptomyces clavuligerus

KM Value [mM]

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

Metals/Ions

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

Natural Substrates/ Products (Substrates)

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

Organism

Organism UniProt Comment Textmining
Streptomyces clavuligerus P42220
-
-
Streptomyces clavuligerus ATCC 27064 P42220
-
-

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
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
-
?
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
-
?
deacetoxycephalosporin C + 2-oxoglutarate + O2
-
Streptomyces clavuligerus deacetylcephalosporin C + succinate + CO2
-
?
deacetoxycephalosporin C + 2-oxoglutarate + O2
-
Streptomyces clavuligerus ATCC 27064 deacetylcephalosporin C + succinate + CO2
-
?

Synonyms

Synonyms Comment Organism
cefF
-
Streptomyces clavuligerus
DACS
-
Streptomyces clavuligerus
deacetylcephalosporin C synthase
-
Streptomyces clavuligerus

Temperature Optimum [°C]

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

Turnover Number [1/s]

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

pH Optimum

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

General Information

General Information Comment Organism
evolution the enzyme belongs to the family of 2-oxoglutarate-dependent oxygenases Streptomyces clavuligerus
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
additional information homology modeling of DACS structure Streptomyces clavuligerus
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]

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