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Literature summary extracted from

  • Dong, Y.; Yan, J.; Du, H.; Chen, M.; Ma, T.; Feng, L.
    Engineering of LadA for enhanced hexadecane oxidation using random- and site-directed mutagenesis (2012), Appl. Microbiol. Biotechnol., 94, 1019-1029.
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.14.14.28
-
 Geobacillus thermodenitrificans

Protein Variants

EC Number Protein Variants Comment Organism
1.14.14.28 A102D hydroxylation activity of purified LadA mutant on hexadecane is 2.1fold higher than that of the wild-type enzyme. Hexadecane degradation rate is 2.3fold higher than that of the wild-type enzyme. A Pseudomonas fluorescens KOB2DELTA1 strain expressing the LadA mutant grows more rapidly with hexadecane than the strain expressing wild-type LadA, confirming the enhanced activity of LadA mutant in vivo. Mutant enzyme with the same size as the wild-type LadA protein. Compared to the wild-type enzyme, the mutant enzyme utilizes a narrower spectrum of n-alkanes, including C16 to C28 Geobacillus thermodenitrificans
1.14.14.28 A102D/F146C/L320V/N376I mutant enzyme completely loses the catalytic activity Geobacillus thermodenitrificans
1.14.14.28 A102D/F146C/N376I mutant enzyme completely loses the catalytic activity Geobacillus thermodenitrificans
1.14.14.28 A102D/L320V mutant enzyme completely loses the catalytic activity Geobacillus thermodenitrificans
1.14.14.28 A102E hydroxylation activity of purified LadA mutant on hexadecane is 2.2fold higher than that of the wild-type enzyme. Hexadecane degradation rate is 2.2fold higher than that of the wild-type enzyme. A Pseudomonas fluorescens KOB2DELTA1 strain expressing the LadA mutant grows more rapidly with hexadecane than the strain expressing wild-type LadA, confirming the enhanced activity of LadA mutant in vivo. Mutant enzyme with the same size as the wild-type LadA protein Geobacillus thermodenitrificans
1.14.14.28 F146C mutant enzyme completely loses the catalytic activity Geobacillus thermodenitrificans
1.14.14.28 F146C/L320V/N376I mutant enzyme completely loses the catalytic activity Geobacillus thermodenitrificans
1.14.14.28 F146C/N376I hydroxylation activity of purified LadA mutant on hexadecane is 2.9fold higher than that of the wild-type enzyme. Hexadecane degradation rate is 2.9fold higher than that of the wild-type enzyme. A Pseudomonas fluorescens KOB2DELTA1 strain expressing the LadA mutant grows more rapidly with hexadecane than the strain expressing wild-type LadA, confirming the enhanced activity of LadA mutant in vivo. Mutant enzyme with the same size as the wild-type LadA protein Geobacillus thermodenitrificans
1.14.14.28 F146E/N376I hydroxylation activity of purified LadA mutant on hexadecane is 2.0fold higher than that of the wild-type enzyme. Hexadecane degradation rate is 2.7fold higher than that of the wild-type enzyme. A Pseudomonas fluorescens KOB2DELTA1 strain expressing the LadA mutant grows more rapidly with hexadecane than the strain expressing wild-type LadA, confirming the enhanced activity of LadA mutant in vivo. Mutant enzyme with the same size as the wild-type LadA protein. Compared to the wild-type enzyme, the mutant enzyme utilizes a narrower spectrum of n-alkanes, including C15 to C28 Geobacillus thermodenitrificans
1.14.14.28 F146N/N376I hydroxylation activity of purified LadA mutant on hexadecane is 3.4fold higher than that of the wild-type enzyme. Hexadecane degradation rate is 3.4fold higher than that of the wild-type enzyme. A Pseudomonas fluorescens KOB2DELTA1 strain expressing the LadA mutant grows more rapidly with hexadecane than the strain expressing wild-type LadA, confirming the enhanced activity of LadA mutant in vivo. The mutant enzyme shows a shift in optimum temperature from 60°C (for the wild-type enzyme) to 75°C. Compared to the wild-type enzyme, the mutant enzyme utilizes a narrower spectrum of n-alkanes, including C15 to C28 Geobacillus thermodenitrificans
1.14.14.28 F146Q/N376I hydroxylation activity of purified LadA mutant on hexadecane is 2.3fold higher than that of the wild-type enzyme. Hexadecane degradation rate is 2.3fold higher than that of the wild-type enzyme. A Pseudomonas fluorescens KOB2DELTA1 strain expressing the LadA mutant grows more rapidly with hexadecane than the strain expressing wild-type LadA, confirming the enhanced activity of LadA mutant in vivo. Mutant enzyme with the same size as the wild-type LadA protein. Compared to the wild-type enzyme, the mutant enzyme utilizes a narrower spectrum of n-alkanes, including C14 to C24 Geobacillus thermodenitrificans
1.14.14.28 F146R/N376I hydroxylation activity of purified LadA mutant on hexadecane is 2.5fold higher than that of the wild-type enzyme. Hexadecane degradation rate is 2.8fold higher than that of the wild-type enzyme. A Pseudomonas fluorescens KOB2DELTA1 strain expressing the LadA mutant grows more rapidly with hexadecane than the strain expressing wild-type LadA, confirming the enhanced activity of LadA mutant in vivo. Mutant enzyme with the same size as the wild-type LadA protein. The mutant enzyme is more heat resistant than wild-type protein, with more than half of the initial activity being retained after incubation at 60°C for 12 h, compared to a 60°C incubation of 4 h or less resulting in the loss of half of the initial activity in the wild-type and F146N/N376I mutant. The mutant enzyme shows a shift in optimum temperature from 60°C (for the wild-type enzyme) to 65°C. Compared to the wild-type enzyme, the mutant enzyme utilizes a narrower spectrum of n-alkanes, including C15 to C24 Geobacillus thermodenitrificans
1.14.14.28 L320A hydroxylation activity of purified LadA mutant on hexadecane is 2.2fold higher than that of the wild-type enzyme. Hexadecane degradation rate is 2.5fold higher than that of the wild-type enzyme. A Pseudomonas fluorescens KOB2DELTA1 strain expressing the LadA mutant grows more rapidly with hexadecane than the strain expressing wild-type LadA, confirming the enhanced activity of LadA mutant in vivo. Mutant enzyme with the same size as the wild-type LadA protein. Compared to the wild-type enzyme, the mutant enzyme utilizes a narrower spectrum of n-alkanes, including C15 to C22 Geobacillus thermodenitrificans
1.14.14.28 L320V hydroxylation activity of purified LadA mutant on hexadecane is 2.5fold higher than that of the wild-type enzyme. Hexadecane degradation rate is 2.4fold higher than that of the wild-type enzyme. A Pseudomonas fluorescens KOB2DELTA1 strain expressing the LadA mutant grows more rapidly with hexadecane than the strain expressing wild-type LadA, confirming the enhanced activity of LadA mutant in vivo. Mutant enzyme with the same size as the wild-type LadA protein Geobacillus thermodenitrificans
1.14.14.28 N376I mutant enzyme completely loses the catalytic activity Geobacillus thermodenitrificans

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.14.14.28 1.4
-
 hexadecane pH 7.5, 60°C, mutant enzyme F146E/N376I Geobacillus thermodenitrificans
1.14.14.28 1.5
-
 hexadecane pH 7.5, 60°C, mutant enzyme A102E Geobacillus thermodenitrificans
1.14.14.28 1.8
-
 hexadecane pH 7.5, 60°C, mutant enzyme A102D Geobacillus thermodenitrificans
1.14.14.28 2
-
 hexadecane pH 7.5, 60°C, mutant enzyme F146R/N376I Geobacillus thermodenitrificans
1.14.14.28 2.7
-
 hexadecane pH 7.5, 60°C, mutant enzyme F146Q/N376I Geobacillus thermodenitrificans
1.14.14.28 2.8
-
 hexadecane pH 7.5, 60°C, mutant enzyme F146C/N376I Geobacillus thermodenitrificans
1.14.14.28 7.1
-
 hexadecane pH 7.5, 60°C, mutant enzyme L320A Geobacillus thermodenitrificans
1.14.14.28 8.9
-
 hexadecane pH 7.5, 60°C, mutant enzyme F146N/N376I Geobacillus thermodenitrificans
1.14.14.28 9.1
-
 hexadecane pH 7.5, 60°C, wild-type enzyme Geobacillus thermodenitrificans
1.14.14.28 11
-
 hexadecane pH 7.5, 60°C, mutant enzyme L320V Geobacillus thermodenitrificans

Organism

EC Number Organism UniProt Comment Textmining
1.14.14.28 Geobacillus thermodenitrificans A4IU28
-
-
1.14.14.28 Geobacillus thermodenitrificans NG80-2 A4IU28
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.14.14.28 wild-type and N-terminal His-tagged fusion proteins Geobacillus thermodenitrificans

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.14.14.28 docosan + FMNH2 + O2
-
 Geobacillus thermodenitrificans 1-docosanol + FMN + H2O
-
 ?
1.14.14.28 docosan + FMNH2 + O2
-
 Geobacillus thermodenitrificans NG80-2 1-docosanol + FMN + H2O
-
 ?
1.14.14.28 dotriacontan + FMNH2 + O2
-
 Geobacillus thermodenitrificans 1-dotriacontanol + FMN + H2O
-
 ?
1.14.14.28 dotriacontan + FMNH2 + O2
-
 Geobacillus thermodenitrificans NG80-2 1-dotriacontanol + FMN + H2O
-
 ?
1.14.14.28 hexacosan + FMNH2 + O2
-
 Geobacillus thermodenitrificans 1-hexacosanol + FMN + H2O
-
 ?
1.14.14.28 hexacosan + FMNH2 + O2
-
 Geobacillus thermodenitrificans NG80-2 1-hexacosanol + FMN + H2O
-
 ?
1.14.14.28 hexadecane + FMNH2 + O2
-
 Geobacillus thermodenitrificans 1-hexadecanol + FMN + H2O
-
 ?
1.14.14.28 hexadecane + FMNH2 + O2
-
 Geobacillus thermodenitrificans NG80-2 1-hexadecanol + FMN + H2O
-
 ?
1.14.14.28 hexatriacontan + FMNH2 + O2
-
 Geobacillus thermodenitrificans 1-hexatriacontanol + FMN + H2O
-
 ?
1.14.14.28 hexatriacontan + FMNH2 + O2
-
 Geobacillus thermodenitrificans NG80-2 1-hexatriacontanol + FMN + H2O
-
 ?
1.14.14.28 octacosan + FMNH2 + O2
-
 Geobacillus thermodenitrificans 1-octacosanol + FMN + H2O
-
 ?
1.14.14.28 octadecane + FMNH2 + O2
-
 Geobacillus thermodenitrificans 1-octadecanol + FMN + H2O
-
 ?
1.14.14.28 pentadecane + FMNH2 + O2
-
 Geobacillus thermodenitrificans 1-pentadecanol + FMN + H2O
-
 ?
1.14.14.28 tetracosan + FMNH2 + O2
-
 Geobacillus thermodenitrificans 1-tetracosanol + FMN + H2O
-
 ?

Subunits

EC Number Subunits Comment Organism
1.14.14.28 homodimer
-
 Geobacillus thermodenitrificans

Synonyms

EC Number Synonyms Comment Organism
1.14.14.28 LADA
-
 Geobacillus thermodenitrificans

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.14.14.28 60
-
 wild-type enzyme Geobacillus thermodenitrificans
1.14.14.28 65
-
 mutant enzyme F146R/N376I Geobacillus thermodenitrificans
1.14.14.28 75
-
 mutant enzyme F146N/N376I Geobacillus thermodenitrificans

Temperature Range [°C]

EC Number Temperature Minimum [°C] Temperature Maximum [°C] Comment Organism
1.14.14.28 40 90 both the wild-type and the mutants are active at temperatures ranging from 40°C to 90°C Geobacillus thermodenitrificans

Temperature Stability [°C]

EC Number Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
1.14.14.28 60
-
 incubation of 4 h or less results in the loss of half of the initial activity in the wild-type and F146N/N376I mutant. Mutant enzyme F146R/N376I retains more than half of the initial activity after incubation for 12 h Geobacillus thermodenitrificans

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.14.14.28 0.02
-
 hexadecane pH 7.5, 60°C, wild-type enzyme Geobacillus thermodenitrificans
1.14.14.28 0.043
-
 hexadecane pH 7.5, 60°C, mutant enzyme F146E/N376I Geobacillus thermodenitrificans
1.14.14.28 0.045
-
 hexadecane pH 7.5, 60°C, mutant enzyme A102D Geobacillus thermodenitrificans
1.14.14.28 0.047
-
 hexadecane pH 7.5, 60°C, mutant enzyme A102E Geobacillus thermodenitrificans
1.14.14.28 0.047
-
 hexadecane pH 7.5, 60°C, mutant enzyme L320A Geobacillus thermodenitrificans
1.14.14.28 0.05
-
 hexadecane pH 7.5, 60°C, mutant enzyme F146Q/N376I Geobacillus thermodenitrificans
1.14.14.28 0.053
-
 hexadecane pH 7.5, 60°C, mutant enzyme L320V Geobacillus thermodenitrificans
1.14.14.28 0.055
-
 hexadecane pH 7.5, 60°C, mutant enzyme F146R/N376I Geobacillus thermodenitrificans
1.14.14.28 0.063
-
 hexadecane pH 7.5, 60°C, mutant enzyme F146C/N376I Geobacillus thermodenitrificans
1.14.14.28 0.073
-
 hexadecane pH 7.5, 60°C, mutant enzyme F146N/N376I Geobacillus thermodenitrificans

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.14.14.28 7
-
 mutant enzyme F146R/N376I Geobacillus thermodenitrificans
1.14.14.28 7.5
-
 wild-type enzyme Geobacillus thermodenitrificans

pH Range

EC Number pH Minimum pH Maximum Comment Organism
1.14.14.28 6 8.8 both the wild-type and the mutants are active at pH values from 6.0 to 8.8 Geobacillus thermodenitrificans

Cofactor

EC Number Cofactor Comment Organism Structure
1.14.14.28 FMN
-
 Geobacillus thermodenitrificans
1.14.14.28 FMNH2
-
 Geobacillus thermodenitrificans

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
1.14.14.28 0.0025
-
 hexadecane pH 7.5, 60°C, wild-type enzyme Geobacillus thermodenitrificans
1.14.14.28 0.0048
-
 hexadecane pH 7.5, 60°C, mutant enzyme L320V Geobacillus thermodenitrificans
1.14.14.28 0.0067
-
 hexadecane pH 7.5, 60°C, mutant enzyme L320A Geobacillus thermodenitrificans
1.14.14.28 0.0082
-
 hexadecane pH 7.5, 60°C, mutant enzyme F146N/N376I Geobacillus thermodenitrificans
1.14.14.28 0.0185
-
 hexadecane pH 7.5, 60°C, mutant enzyme F146Q/N376I Geobacillus thermodenitrificans
1.14.14.28 0.022
-
 hexadecane pH 7.5, 60°C, mutant enzyme F146C/N376I Geobacillus thermodenitrificans
1.14.14.28 0.0245
-
 hexadecane pH 7.5, 60°C, mutant enzyme A102D Geobacillus thermodenitrificans
1.14.14.28 0.0295
-
 hexadecane pH 7.5, 60°C, mutant enzyme F146R/N376I Geobacillus thermodenitrificans
1.14.14.28 0.031
-
 hexadecane pH 7.5, 60°C, mutant enzyme F146E/N376I Geobacillus thermodenitrificans
1.14.14.28 0.0315
-
 hexadecane pH 7.5, 60°C, mutant enzyme A102E Geobacillus thermodenitrificans