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1.14.20.1: deacetoxycephalosporin-C synthase

This is an abbreviated version!
For detailed information about deacetoxycephalosporin-C synthase, go to the full flat file.

Word Map on EC 1.14.20.1

Reaction

Penicillin N
+
2-oxoglutarate
 +
O2
=
deacetoxycephalosporin C
 +
succinate
 +
CO2
 +
H2O

Synonyms

acDAOC/DACS, cefE, cefEF, Cephalosporin biosynthesis expandase/hydroxylase, DAOC synthase, DAOC/DAC synthase, DAOC/DACS, DAOCS, deacetoxy/deacetylcephalosporin C synthase, deacetoxycephalosporin C synthase, deacetoxycephalosporin-C synthase, deacetoxycephalosporin-C synthetase, deacetoxycephalosporin/deacetylcephalosporin C synthase, expandase, expendase, penicillin N expandase, scDAOCS

ECTree

     1 Oxidoreductases
         1.14 Acting on paired donors, with incorporation or reduction of molecular oxygen
             1.14.20 With 2-oxoglutarate as one donor, and the other dehydrogenated
                1.14.20.1 deacetoxycephalosporin-C synthase

Engineering

Engineering on EC 1.14.20.1 - deacetoxycephalosporin-C synthase

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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
DELTA310/M306I
site-directed mutagenesis, mutant selectively catalyzed the ring expansion with kinetics similar to the wild-type enzyme
DELTA310/N305L/M306I
site-directed mutagenesis, mutant selectively catalyzed the ring expansion, and shows a lower Km value than the wild-type enzyme and about 3fold improved kinetic parameters
M306I
N305L
R307L
-
activities comparable to wild-type
R308A
-
site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme
R308C
-
site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme
R308D
-
site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme
R308E
-
site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme
R308F
-
site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme
R308G
-
site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme
R308H
-
site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme
R308I
-
site-directed mutagenesis, the mutant shows significant improvement in the ability to convert penicillin analogues compared to the wild-type enzyme. The mutant shows the highest reactivity for penicillin G, with 3fold increase in kcat/Km ratio and 7fold increase in relative activity
R308K
-
site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme
R308L
R308M
-
site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme
R308N
-
site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme
R308P
-
site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme
R308Q
-
site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme
R308S
-
site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme
R308T
-
site-directed mutagenesis, the mutant shows significant improvement in the ability to convert penicillin analogues compared to the wild-type enzyme
R308V
-
site-directed mutagenesis, the mutant shows significant improvement in the ability to convert penicillin analogues compared to the wild-type enzyme
R308W
-
site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme
R308Y
-
site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme
W82S
44% of wild-type ring expansion activity, 18% of wild-type hydroxylation activity
A106T
80% relative activity with 1 mM substrate and 170% relative activity with 10 mM penicillin G compared to wild-type activity detected with 1 mM penicillin G, 1.5fold reduction in Km, penicillin G as substrate
A106T/C155Y
epPCR random mutagenesis
A106T/M188V
epPCR random mutagenesis
A11V/T91A/C281Y/I305L
220% relative activity with 1 mM and 200% relative activity with 10 mM substrate penicillin G compared to wild-type activity detected with 1 mM penicillin G
A61E
-
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type
C155Y
C155Y/Y184H/S261M/V275I/C281Y/I305M
site-directed mutagenesis, the mutant shows 87fold increased kcat/Km for penicillin G compared to the wild-type
C155Y/Y184H/V275I/C281Y
C281F
substrate ampicillin, 191%, penicillin G, 264%, phenethicillin, 300%, and carbenicillin, 518% of wild-type activity
C281R
activity similar to wild-type enzyme
C281Y
C281Y/I305M
C281Y/N304A
substrate ampicillin, 294%, penicillin G, 383%, phenethicillin, 714%, and carbenicillin, 911% of wild-type activity
C281Y/N304K
substrate ampicillin, 376%, penicillin G, 428%, phenethicillin, 948%, and carbenicillin, 1180% of wild-type activity
C281Y/N304L
substrate ampicillin, 330%, penicillin G, 211%, phenethicillin, 471%, and carbenicillin, 1001% of wild-type activity
C281Y/N304M
substrate ampicillin, 225%, penicillin G, 277%, phenethicillin, 428%, and carbenicillin, 485% of wild-type activity
C281Y/N304R
C281Y/R306L
substrate ampicillin, 394%, penicillin G, 191%, phenethicillin, 555%, and carbenicillin, 1010% of wild-type activity
C281Y/R307L
substrate ampicillin, 274%, penicillin G, 334%, phenethicillin, 520%, and carbenicillin, 786% of wild-type activity
C37S
-
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type
D107G
activity similar to wild-type enzyme
D107G/L277Q
epPCR random mutagenesis
D185L
-
no detectable ring expansion activity
D53H/C281Y
epPCR random mutagenesis
DELTAI305-310
mutant truncated by six residues, reduced turnover number for penicillin N compared to wild-type enzyme, lower KM-value for penicillin N compared to wild-type enzyme
DELTAK310-314
mutant truncated by five residues, increased turnover number for penicillin N compared to wild-type enzyme, lower KM-value for penicillin N compared to wild-type enzyme, lower Km-value for penicillin G compared to wild-type enzyme, slightly higher turnover number for penicillin G compared to wild-type enzyme
DELTAR306-310
mutant truncated by five residues, reduced turnover number for penicillin N compared to wild-type enzyme, lower KM-value for penicillin N compared to wild-type enzyme
DELTAR307-310
mutant in which residues 307-310 are excised, reduced turnover number for penicillin N compared to wild-type enzyme, higher KM-value for penicillin N compared to wild-type enzyme, increased KM-value for penicillin G compared to wild-type enzyme, increased turnover-number for penicillin G compared to wild-type enzyme
E144K/M188I/A198T/V275I/C281Y
160% relative activity with 1 mM and 200% relative activity with 10 mM substrate penicillin G compared to wild-type activity detected with 1 mM penicillin G
G300V
H183L
-
no detectable ring expansion activity
H243L
-
no detectable ring expansion activity
H244Q
I305L
I305M
I305M/S261M
-
site-directed mutagenesis, the mutant shows increased activity with penicillin G compared to the wild-type enzyme
I305M/T213V
-
site-directed mutagenesis, the mutant shows increased activity with penicillin G compared to the wild-type enzyme
I305M/T213V/M73T
-
site-directed mutagenesis, the mutant shows increased activity with penicillin G compared to the wild-type enzyme
I305M/T213V/S261M
-
site-directed mutagenesis, the mutant shows increased activity with penicillin G compared to the wild-type enzyme
L277Q
L277Q/I305M
610% relative activity with 1 mM and 520% relative activity with 10 mM substrate penicillin G compared to wild-type activity detected with 1 mM penicillin G
M188I
90% relative activity with 1 mM and 190% relative activity with 10 mM substrate penicillin G compared to wild-type activity detected with 1 mM penicillin G, epPCR random mutagenesis
M188V
M188V/V275I/G300V
440% relative activity with 1 mM and 310% relative activity with 10 mM substrate penicillin G compared to wild-type activity detected with 1 mM penicillin G
M73T/C155Y/Y184H/T213V/V275I/C281Y/I305M
site-directed mutagenesis, the mutant shows 22fold increased specific activity and 81fold increased kcat/Km for penicillin G compared to the wild-type
M73T/C281Y
610% relative activity with 1 mM and 530% relative activity with 10 mM substrate penicillin G compared to wild-type activity detected with 1 mM penicillin G
M73T/G300V/I305L
640% relative activity with 1 mM and 380% relative activity with 10 mM substrate penicillin G compared to wild-type activity detected with 1 mM penicillin G
M73T/I118V/A140V/H244Q/C281Y
450% relative activity with 1 mM and 350% relative activity with 10 mM substrate penicillin G compared to wild-type activity detected with 1 mM penicillin G
M73T/I277Q
350% relative activity with 1 mM and 350% relative activity with 10 mM substrate penicillin G compared to wild-type activity detected with 1 mM penicillin G
M73T/P145L
epPCR random mutagenesis
N304A
N304A/R306L
substrate ampicillin, 214%, penicillin G, 114%, phenethicillin, 319%, and carbenicillin, 393% of wild-type activity
N304K
N304K/R306L
substrate ampicillin, 245%, penicillin G, 80%, phenethicillin, 380%, and carbenicillin, 482% of wild-type activity
N304L
N304L/R306L
substrate ampicillin, 145%, penicillin G, 40%, phenethicillin, 185%, and carbenicillin, 218% of wild-type activity
N304M
substrate ampicillin, 120%, penicillin G,92%, phenethicillin, 127%, and carbenicillin, 161% of wild-type activity
N304M/R306L
substrate ampicillin, 194%, penicillin G, 72%, phenethicillin, 245%, and carbenicillin, 232% of wild-type activity
N304R
N304R/R306L
substrate ampicillin, 256%, penicillin G, 92%, phenethicillin, 344%, and carbenicillin, 514% of wild-type activity
Q126M
R135Q/C155Y/R179Q/M188V/I305M
360% relative activity with 1 mM and 290% relative activity with 10 mM substrate penicillin G compared to wild-type activity detected with 1 mM penicillin G
R160L
mutation abolishes the activity of scDAOCS indicating the importance of these residue in binding the penicillin substrate
R160Q
-
more than 95% loss of activity
R258A
R258F
R258H
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
R258L
R258Q
R266L
R266Q
-
2-oxoglutarate conversion is very low and the same whether penicillin N, penicillin G or no penicillin substrate is present
R306A
substrate ampicillin, 46%, penicillin G, 37%, phenethicillin, 30%, and carbenicillin, 27% of wild-type activity
R306C
substrate ampicillin, 18%, penicillin G, 13%, phenethicillin, 11%, of wild-type activity
R306D
substrate ampicillin, 8%, penicillin G, 4%, phenethicillin, 4%, of wild-type activity
R306E
substrate ampicillin, 8%, penicillin G, 7%, phenethicillin, 6%,of wild-type activity
R306F
substrate ampicillin, 55%, penicillin G, 41%, phenethicillin, 44%, and carbenicillin, 39% of wild-type activity
R306G
substrate ampicillin, 21%, penicillin G, 27%, phenethicillin, 19%, of wild-type activity
R306H
substrate ampicillin, 16%, penicillin G, 10%, phenethicillin, 8%, of wild-type activity
R306I
R306K
substrate ampicillin, 50%, penicillin G, 53%, phenethicillin, 45%, and carbenicillin, 32% of wild-type activity
R306L
R306M
R306N
substrate ampicillin, 16%, penicillin G, 14%, phenethicillin, 12%, and carbenicillin, 16% of wild-type activity
R306P
R306Q
substrate ampicillin, 62%, penicillin G, 67%, phenethicillin, 54%, and carbenicillin, 62% of wild-type activity
R306S
substrate ampicillin, 39%, penicillin G, 37%, phenethicillin, 27%, and carbenicillin, 30% of wild-type activity
R306T
substrate ampicillin, 40%, penicillin G, 52%, phenethicillin, 32%, and carbenicillin, 34% of wild-type activity
R306V
substrate ampicillin, 34%, penicillin G, 28%, phenethicillin, 36%, and carbenicillin, 32% of wild-type activity
R306W
substrate ampicillin, 90%, penicillin G, 61%, phenethicillin, 82%, and carbenicillin, 83% of wild-type activity
R306Y
substrate ampicillin, 70%, penicillin G, 59%, phenethicillin, 61%, and carbenicillin, 60% of wild-type activity
R307L
R307Q
-
mutation enhances penicillin N conversion compared to the level of wild-type enzyme, turnover number for penicillin N is increased, no enhancement in activity with penicillin G as substrate. Mutation increase the Km-value by 10fold, but has little effect on the turnover number for penicillin G
R74I
-
turnover number and Km-values are similar to that for wild-type enzyme. 2-Oxoglutarate conversion is significantly stimulated in presence of penicillin N and penicillin G compared to wild type enzyme. Penicillin oxidation is reduced relative to the wild type enzyme
R74L
mutation abolishes the activity of scDAOCS indicating the importance of these residue in binding the penicillin substrate
R74Q
-
turnover number and Km-values are similar to that for wild-type enzyme. 2-Oxoglutarate conversion is significantly stimulated in presence of penicillin N and penicillin G compared to wild type enzyme. Penicillin oxidation is reduced relative to the wild type enzyme
R74Q/R266I
-
2-oxoglutarate conversion is not stimulated by penicillin substrates
R74Q/R266Q
-
2-oxoglutarate conversion is not stimulated by penicillin substrates
R75I/D270G
-
2-oxoglutarate conversion is not stimulated by penicillin substrates
R75Q
-
turnover number and Km-values are similar to that for wild-type enzyme. 2-Oxoglutarate conversion is significantly stimulated in presence of penicillin N compared to wild type enzyme
S261A
S261I
-
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type
S261L
-
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type
S261M
S261V
-
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type
S59T
-
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type
T213V
T42A
-
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type
V245H
-
site-directed mutagenesis, inactive mutant
V245K
-
site-directed mutagenesis, inactive mutant
V245R
-
site-directed mutagenesis, inactive mutant
V275I
V275I/C281Y
260% relative activity with 1 mM and 310% relative activity with 10 mM substrate penicillin G compared to wild-type activity detected with 1 mM penicillin G
V275I/C281Y/G300V
620% relative activity with 1 mM and 380% relative activity with 10 mM substrate penicillin G compared to wild-type activity detected with 1 mM penicillin G
V275I/C281Y/I305M
-
site-directed mutagenesis, exchange of residue surrounding the substrate, over 32fold increased activity compared to the wild-type enzyme
V275I/I305L
310% relative activity with 1 mM and 330% relative activity with 10 mM substrate penicillin G compared to wild-type activity detected with 1 mM penicillin G
V275I/I305M
V275I/N304A
substrate ampicillin, 265%, penicillin G, 233%, phenethicillin, 330%, and carbenicillin,721% of wild-type activity
V275I/N304K
substrate ampicillin, 334%, penicillin G, 294%, phenethicillin, 635%, and carbenicillin, 867% of wild-type activity
V275I/N304L
substrate ampicillin, 225%, penicillin G, 141%, phenethicillin, 245%, and carbenicillin, 524% of wild-type activity
V275I/N304M
substrate ampicillin, 160%, penicillin G, 174%, phenethicillin, 261%, and carbenicillin, 230% of wild-type activity
V275I/N304R
substrate ampicillin, 360%, penicillin G, 381%, phenethicillin, 698%, and carbenicillin,814% of wild-type activity
V275I/R306L
substrate ampicillin, 343%, penicillin G, 168%, phenethicillin, 379%, and carbenicillin, 665% of wild-type activity
V275I/R307L
substrate ampicillin, 172%, penicillin G, 169%, phenethicillin, 238%, and carbenicillin, 333% of wild-type activity
V275L
substrate ampicillin, 85%, penicillin G, 130%, phenethicillin, 99%, and carbenicillin, 113% of wild-type activity
V51A
-
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type
Y184A
-
site-directed mutagenesis, the mutant shows activity increased to 143.9% with penicillin G compared to the wild-type
Y184F
-
site-directed mutagenesis, inactive mutant
Y184H
Y184H/C281R
epPCR random mutagenesis
Y184H/H244Q/T259I/L277Q
320% relative activity with 1 mM and 310% relative activity with 10 mM substrate penicillin G compared to wild-type activity detected with 1 mM penicillin G
Y184H/M188I/C281Y/I305L
610% relative activity with 1 mM and 460% relative activity with 10 mM substrate penicillin G compared to wild-type activity detected with 1 mM penicillin G
Y184I
-
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type
Y184L
-
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type
Y184M
-
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type
Y184V
-
site-directed mutagenesis, inactive mutant
C155Y/Y184H/S261M/V275I/C281Y/I305M
-
site-directed mutagenesis, the mutant shows 87fold increased kcat/Km for penicillin G compared to the wild-type
-
C155Y/Y184H/V275I/C281Y
-
site-directed mutagenesis, quaternary mutant from error-prone PCR, the mutant shows 41fold increased kcat/Km for penicillin G compared to the wild-type
-
C37S
-
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type
-
M73T/C155Y/Y184H/T213V/V275I/C281Y/I305M
-
site-directed mutagenesis, the mutant shows 22fold increased specific activity and 81fold increased kcat/Km for penicillin G compared to the wild-type
-
T42A
-
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type
-
V51A
-
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type
-
Y184A
-
site-directed mutagenesis, the mutant shows activity increased to 143.9% with penicillin G compared to the wild-type
-
R258L
-
mutant have the same catalytic activity as the R258Q mutant when 2-oxo-4-methylpentanoate is used as a co-substrate
-
R258Q
-
mutation in scDAOCS almost abolishes the oxidation of both penicillin N and penicillin G, aliphatic 2-oxoacids (2-oxo-4-methylpentanoate and 2-oxo-3-methylbutanoate), which can not replace the function of 2-oxoglutarate for the wild-type enzyme, are able to rescue the catalytic activity of the R258Q mutant to the level observed for the wild-type enzyme
-
R266L
-
mutation abolishes the activity of scDAOCS indicating the importance of these residue in binding the penicillin substrate
-
R306L
-
mutation improves the conversion activity of scDAOCS
-
R74L
-
mutation abolishes the activity of scDAOCS indicating the importance of these residue in binding the penicillin substrate
-
additional information