BRENDA - Enzyme Database

Redesign of soluble fatty acid desaturases from plants from altered substrate specificity and double bond position

Cahoon, E.B.; Lindqvist, Y.; Schneider, G.; Shanklin, J.; Proc. Natl. Acad. Sci. USA 94, 4872-4877 (1997)

Data extracted from this reference:

Cloned(Commentary)
EC Number
Commentary
Organism
1.14.19.3
expression of wild-type, A181T/A188G/Y189F/S205N/L206T/G207A, A188G/Y189F, A181T/A200F and A181T/A200F/S205N/L206T/G207A mutant enzyme in Escherichia coli
Thunbergia alata
1.14.19.26
expression of wild-type and mutant enzymes in Escherichia coli
Thunbergia alata
Engineering
EC Number
Amino acid exchange
Commentary
Organism
1.14.19.2
L118F/P179I
15fold higher activity with palmitoyl-[acyl-carrier protein] than wild-type enzyme, low DELTA10 desaturase activity
Ricinus communis
1.14.19.3
A181T/A188G/Y189F/S205N/L206T/G207A
compared to wild-type: reduced DELTA6 desaturase activity with palmitoyl-[acyl-carrier protein] as substrate, strong DELTA9 desaturase activity with stearoyl-[acyl-carrier protein] as substrate, exhibits DELTA9 desaturase activity with palmitoyl-[acyl-carrier protein] as substrate
Thunbergia alata
1.14.19.3
A181T/A200F
increase in DELTA6 desaturase activity with palmitoyl-[acyl-carrier protein] as substrate, strong DELTA9 desaturase activity with stearoyl-[acyl-carrier protein] as substrate
Thunbergia alata
1.14.19.3
A181T/A200F/S205N/L206T/G207A
reduced DELTA6 desaturase activity with palmitoyl-[acyl-carrier protein] as substrate, very low DELTA9 desaturase activity, no DELTA6 desaturase activity with stearoyl-[acyl-carrier protein] as substrate
Thunbergia alata
1.14.19.3
A188G/Y189F
reduced DELTA6 desaturase activity with palmitoyl-[acyl-carrier protein] as substrate
Thunbergia alata
1.14.19.26
A181T/A188G/Y189F/S205N/L206T/G207A
the mutation yields an enzyme that functions primarily as a DELTA9-18:0-[acyl-carrier protein] desaturase. This enzyme displays only DELTA9 desaturase activity with 18:0-[acyl-carrier protein] and is nearly 4-fold more active with this substrate than with 16:0-[acyl-carrier protein]. Like mutant A181TyA200F, this enzyme retains DELTA6 desaturase activity with 16:0-[acyl-carrier protein]
Thunbergia alata
1.14.19.26
A181T/A200F
the mutation gives rise to an enzyme that catalyzed primarily the DELTA9 desaturation of 18:0-[acyl-carrier protein], but functions as a DELTA6 desaturase with 16:0-[acyl-carrier protein]
Thunbergia alata
1.14.19.26
A181T/A200F/S205N/L206T/G207A
replacement of specific amino acid residues in a DELTA6-palmitoyl (16:0)-[acyl-carrier protein] desaturase with their equivalents from a DELTA9-stearoyl (18:0)-[acyl-carrier protein] desaturase. The A181T/A200F/S205N/L206T/G207A mutant enzyme is functions principally as a DELTA9-18:0-[acyl-carrier protein] desaturase
Thunbergia alata
1.14.19.26
A188G/Y189F
mutant with broadened fatty acid chain-length specificity
Thunbergia alata
1.14.19.26
additional information
replacement of specific amino acid residues in a DELTA6-palmitoyl (16:0)-[acyl-carrier protein] desaturase with their equivalents from a DELTA9-stearoyl (18:0)-[acyl-carrier protein] desaturase, mutant enzymes are identified that have altered fatty acid chain-length specificities or that can insert double bonds into either the DELTA6 or DELTA9 positions of 16:0- and 18:0-[acyl-carrier protein]
Thunbergia alata
KM Value [mM]
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
1.14.19.26
additional information
-
palmitoyl-[acyl-carrier protein]
Km-values determined for the wild-type enzyme and mutant A188G/Y189F are in the range of 0.2 to 0.6 mM with both 16:0- and 18:0-[acyl-carrier protein], suggesting that, at least in the case of these enzymes, changes in the substrate binding properties can be discounted as an underlying cause of differences in activities
Thunbergia alata
Natural Substrates/ Products (Substrates)
EC Number
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
1.14.19.26
palmitoyl-[acyl-carrier protein] + 2 reduced ferredoxin [iron-sulfur] cluster + O2 + 2 H+
Thunbergia alata
-
(Z)-hexadec-6-enoyl-[acyl-carrier protein] + 2 oxidized ferredoxin [iron-sulfur] cluster + 2 H2O
-
-
?
Organism
EC Number
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
1.14.19.2
Ricinus communis
P22337
-
-
1.14.19.3
Thunbergia alata
-
black-eyed Susan vine
-
1.14.19.26
Thunbergia alata
Q41510
-
-
Purification (Commentary)
EC Number
Commentary
Organism
1.14.19.3
recombinant enzyme
Thunbergia alata
1.14.19.26
-
Thunbergia alata
Specific Activity [micromol/min/mg]
EC Number
Specific Activity Minimum [µmol/min/mg]
Specific Activity Maximum [µmol/min/mg]
Commentary
Organism
1.14.19.3
0.1
-
substrate palmitoyl-[acyl-carrier protein]
Thunbergia alata
Substrates and Products (Substrate)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
1.14.19.3
palmitoyl-[acyl-carrier protein] + reduced acceptor + O2
-
438436
Thunbergia alata
6-hexadecenoyl-[acyl-carrier protein] + acceptor + H2O
-
438436
Thunbergia alata
?
1.14.19.3
stearoyl-[acyl-carrier protein] + reduced acceptor + O2
-
438436
Thunbergia alata
6-octadecenoyl-[acyl-carrier protein] + 9-octadecenoyl-[acyl-carrier protein]acceptor + H2O
ratio of activity: 2/1
438436
Thunbergia alata
?
1.14.19.26
palmitoyl-[acyl-carrier protein] + 2 reduced ferredoxin [iron-sulfur] cluster + O2 + 2 H+
-
438436
Thunbergia alata
(Z)-hexadec-6-enoyl-[acyl-carrier protein] + 2 oxidized ferredoxin [iron-sulfur] cluster + 2 H2O
-
-
-
?
Cofactor
EC Number
Cofactor
Commentary
Organism
Structure
1.14.19.2
Ferredoxin
-
Ricinus communis
1.14.19.3
Ferredoxin
-
Thunbergia alata
Cloned(Commentary) (protein specific)
EC Number
Commentary
Organism
1.14.19.3
expression of wild-type, A181T/A188G/Y189F/S205N/L206T/G207A, A188G/Y189F, A181T/A200F and A181T/A200F/S205N/L206T/G207A mutant enzyme in Escherichia coli
Thunbergia alata
1.14.19.26
expression of wild-type and mutant enzymes in Escherichia coli
Thunbergia alata
Cofactor (protein specific)
EC Number
Cofactor
Commentary
Organism
Structure
1.14.19.2
Ferredoxin
-
Ricinus communis
1.14.19.3
Ferredoxin
-
Thunbergia alata
Engineering (protein specific)
EC Number
Amino acid exchange
Commentary
Organism
1.14.19.2
L118F/P179I
15fold higher activity with palmitoyl-[acyl-carrier protein] than wild-type enzyme, low DELTA10 desaturase activity
Ricinus communis
1.14.19.3
A181T/A188G/Y189F/S205N/L206T/G207A
compared to wild-type: reduced DELTA6 desaturase activity with palmitoyl-[acyl-carrier protein] as substrate, strong DELTA9 desaturase activity with stearoyl-[acyl-carrier protein] as substrate, exhibits DELTA9 desaturase activity with palmitoyl-[acyl-carrier protein] as substrate
Thunbergia alata
1.14.19.3
A181T/A200F
increase in DELTA6 desaturase activity with palmitoyl-[acyl-carrier protein] as substrate, strong DELTA9 desaturase activity with stearoyl-[acyl-carrier protein] as substrate
Thunbergia alata
1.14.19.3
A181T/A200F/S205N/L206T/G207A
reduced DELTA6 desaturase activity with palmitoyl-[acyl-carrier protein] as substrate, very low DELTA9 desaturase activity, no DELTA6 desaturase activity with stearoyl-[acyl-carrier protein] as substrate
Thunbergia alata
1.14.19.3
A188G/Y189F
reduced DELTA6 desaturase activity with palmitoyl-[acyl-carrier protein] as substrate
Thunbergia alata
1.14.19.26
A181T/A188G/Y189F/S205N/L206T/G207A
the mutation yields an enzyme that functions primarily as a DELTA9-18:0-[acyl-carrier protein] desaturase. This enzyme displays only DELTA9 desaturase activity with 18:0-[acyl-carrier protein] and is nearly 4-fold more active with this substrate than with 16:0-[acyl-carrier protein]. Like mutant A181TyA200F, this enzyme retains DELTA6 desaturase activity with 16:0-[acyl-carrier protein]
Thunbergia alata
1.14.19.26
A181T/A200F
the mutation gives rise to an enzyme that catalyzed primarily the DELTA9 desaturation of 18:0-[acyl-carrier protein], but functions as a DELTA6 desaturase with 16:0-[acyl-carrier protein]
Thunbergia alata
1.14.19.26
A181T/A200F/S205N/L206T/G207A
replacement of specific amino acid residues in a DELTA6-palmitoyl (16:0)-[acyl-carrier protein] desaturase with their equivalents from a DELTA9-stearoyl (18:0)-[acyl-carrier protein] desaturase. The A181T/A200F/S205N/L206T/G207A mutant enzyme is functions principally as a DELTA9-18:0-[acyl-carrier protein] desaturase
Thunbergia alata
1.14.19.26
A188G/Y189F
mutant with broadened fatty acid chain-length specificity
Thunbergia alata
1.14.19.26
additional information
replacement of specific amino acid residues in a DELTA6-palmitoyl (16:0)-[acyl-carrier protein] desaturase with their equivalents from a DELTA9-stearoyl (18:0)-[acyl-carrier protein] desaturase, mutant enzymes are identified that have altered fatty acid chain-length specificities or that can insert double bonds into either the DELTA6 or DELTA9 positions of 16:0- and 18:0-[acyl-carrier protein]
Thunbergia alata
KM Value [mM] (protein specific)
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
1.14.19.26
additional information
-
palmitoyl-[acyl-carrier protein]
Km-values determined for the wild-type enzyme and mutant A188G/Y189F are in the range of 0.2 to 0.6 mM with both 16:0- and 18:0-[acyl-carrier protein], suggesting that, at least in the case of these enzymes, changes in the substrate binding properties can be discounted as an underlying cause of differences in activities
Thunbergia alata
Natural Substrates/ Products (Substrates) (protein specific)
EC Number
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
1.14.19.26
palmitoyl-[acyl-carrier protein] + 2 reduced ferredoxin [iron-sulfur] cluster + O2 + 2 H+
Thunbergia alata
-
(Z)-hexadec-6-enoyl-[acyl-carrier protein] + 2 oxidized ferredoxin [iron-sulfur] cluster + 2 H2O
-
-
?
Purification (Commentary) (protein specific)
EC Number
Commentary
Organism
1.14.19.3
recombinant enzyme
Thunbergia alata
1.14.19.26
-
Thunbergia alata
Specific Activity [micromol/min/mg] (protein specific)
EC Number
Specific Activity Minimum [µmol/min/mg]
Specific Activity Maximum [µmol/min/mg]
Commentary
Organism
1.14.19.3
0.1
-
substrate palmitoyl-[acyl-carrier protein]
Thunbergia alata
Substrates and Products (Substrate) (protein specific)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
1.14.19.3
palmitoyl-[acyl-carrier protein] + reduced acceptor + O2
-
438436
Thunbergia alata
6-hexadecenoyl-[acyl-carrier protein] + acceptor + H2O
-
438436
Thunbergia alata
?
1.14.19.3
stearoyl-[acyl-carrier protein] + reduced acceptor + O2
-
438436
Thunbergia alata
6-octadecenoyl-[acyl-carrier protein] + 9-octadecenoyl-[acyl-carrier protein]acceptor + H2O
ratio of activity: 2/1
438436
Thunbergia alata
?
1.14.19.26
palmitoyl-[acyl-carrier protein] + 2 reduced ferredoxin [iron-sulfur] cluster + O2 + 2 H+
-
438436
Thunbergia alata
(Z)-hexadec-6-enoyl-[acyl-carrier protein] + 2 oxidized ferredoxin [iron-sulfur] cluster + 2 H2O
-
-
-
?