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Literature summary extracted from

  • Molle, V.; Gulten, G.; Vilcheze, C.; Veyron-Churlet, R.; Zanella-Cleon, I.; Sacchettini, J.; Jacobs Jr, W.; Kremer, L.
    Phosphorylation of InhA inhibits mycolic acid biosynthesis and growth of Mycobacterium tuberculosis (2010), Mol. Microbiol., 78, 1591-1605 .
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.3.1.9 the inhA gene was cloned into the pMK1 mycobacterial expression vector under the control of the strong promoter hsp60. The resulting construct is used to transform Mycobacterium bovis BCG Pasteur in order to allow over-production of recombinant His-tagged InhA Mycobacterium tuberculosis
1.3.1.118 the inhA gene is cloned into the pMK1 mycobacterial expression vector under the control of the strong promoter hsp60. The resulting construct is used to transform Mycobacterium bovis BCG Pasteur in order to allow overproduction of recombinant His-tagged InhA Mycobacterium tuberculosis

Protein Variants

EC Number Protein Variants Comment Organism
1.3.1.9 T266A phosphoablative mutant with activity similar to wild-type enzyme Mycobacterium tuberculosis
1.3.1.9 T266D phosphomimetic mutant with strongly reduced activity (31.4% compared to wild-type enzyme), introduction of inhA_T266D fails to complement growth and mycolic acid defects of an inhA-thermosensitive Mycobacterium smegmatis strain, in a similar manner to what is observed following isoniazid treatment Mycobacterium tuberculosis
1.3.1.9 T266E phosphomimetic mutant strongly reduced activity (29.5% compared to wild-type enzyme), introduction of inhA_T266E fails to complement growth and mycolic acid defects of an inhA-thermosensitive Mycobacterium smegmatis strain, in a similar manner to what is observed following isoniazid treatment Mycobacterium tuberculosis
1.3.1.118 T266A phosphoablative mutant with activity similar to wild-type enzyme Mycobacterium tuberculosis
1.3.1.118 T266D phosphomimetic mutant with strongly reduced activity (31.4% compared to wild-type enzyme), introduction of inhA_T266D fails to complement growth and mycolic acid defects of an inhA-thermosensitive Mycobacterium smegmatis strain, in a similar manner to what is observed following isoniazid treatment Mycobacterium tuberculosis
1.3.1.118 T266E phosphomimetic mutant with strongly reduced activity (29.5% compared to wild-type enzyme), introduction of inhA_T266E fails to complement growth and mycolic acid defects of an inhA-thermosensitive Mycobacterium smegmatis strain, in a similar manner to what is observed following isoniazid treatment Mycobacterium tuberculosis

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.3.1.9 0.0196
-
 trans-2-dodecenoyl-CoA pH 7.5, 25°C, mutant enzyme T266D Mycobacterium tuberculosis
1.3.1.9 0.0203
-
 trans-2-dodecenoyl-CoA pH 7.5, 25°C, mutant enzyme T266E Mycobacterium tuberculosis
1.3.1.9 0.0409
-
 trans-2-dodecenoyl-CoA pH 7.5, 25°C, wild-type enzyme Mycobacterium tuberculosis
1.3.1.9 0.0523
-
 trans-2-dodecenoyl-CoA pH 7.5, 25°C, mutant enzyme T266A Mycobacterium tuberculosis
1.3.1.118 0.0196
-
 trans-2-dodecenoyl-CoA pH 7.5, 25°C, mutant enzyme T266D Mycobacterium tuberculosis
1.3.1.118 0.0203
-
 trans-2-dodecenoyl-CoA pH 7.5, 25°C, mutant enzyme T266E Mycobacterium tuberculosis
1.3.1.118 0.0409
-
 trans-2-dodecenoyl-CoA pH 7.5, 25°C, wild-type enzyme Mycobacterium tuberculosis
1.3.1.118 0.0523
-
 trans-2-dodecenoyl-CoA pH 7.5, 25°C, mutant enzyme T266A Mycobacterium tuberculosis

Organism

EC Number Organism UniProt Comment Textmining
1.3.1.9 Mycobacterium tuberculosis P9WGR1
-
-
1.3.1.9 Mycobacterium tuberculosis ATCC 25618 P9WGR1
-
-
1.3.1.118 Mycobacterium tuberculosis P9WGR1
-
-
1.3.1.118 Mycobacterium tuberculosis ATCC 25618 P9WGR1
-
-

Posttranslational Modification

EC Number Posttranslational Modification Comment Organism
1.3.1.9 phosphoprotein InhA is phosphorylated in vitro by multiple Ser/Thr kinases on residue Thr266.. Activity of InhA is controlled via phosphorylation. Thr266 is the unique kinase phosphoacceptor, both in vitro and in vivo. The physiological relevance of Thr266 phosphorylation is demonstrated using inhA phosphoablative (T266A) or phosphomimetic (T266D/E) mutants. Enoyl reductase activity is severely impaired in the mimetic mutants in vitro, as a consequence of a reduced binding affinity to NADH. Introduction of inhA_T266D/E fails to complement growth and mycolic acid defects of an inhA-thermosensitive Mycobacterium smegmatis strain, in a similar manner to what is observed following isoniazid treatment. Phosphorylation of InhA may represent an unusual mechanism that allows Mycobacterium tuberculosis to regulate its mycolic acid content, thus offering a new approach to future anti-tuberculosis drug development Mycobacterium tuberculosis
1.3.1.118 phosphoprotein InhA is phosphorylated in vitro by multiple Ser/Thr kinases on residue Thr266.. Activity of InhA is controlled via phosphorylation. Thr266 is the unique kinase phosphoacceptor, both in vitro and in vivo. The physiological relevance of Thr266 phosphorylation is demonstrated using inhA phosphoablative (T266A) or phosphomimetic (T266D/E) mutants. Enoyl reductase activity is severely impaired in the mimetic mutants in vitro, as a consequence of a reduced binding affinity to NADH. Introduction of inhA_T266D/E fails to complement growth and mycolic acid defects of an inhA-thermosensitive Mycobacterium smegmatis strain, in a similar manner to what is observed following isoniazid treatment. Phosphorylation of InhA may represent an unusual mechanism that allows Mycobacterium tuberculosis to regulate its mycolic acid content, thus offering a new approach to future anti-tuberculosis drug development Mycobacterium tuberculosis

Purification (Commentary)

EC Number Purification (Comment) Organism
1.3.1.9
-
 Mycobacterium tuberculosis
1.3.1.118
-
 Mycobacterium tuberculosis

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.3.1.9 trans-2-dodecenoyl-CoA + NADH + H+
-
 Mycobacterium tuberculosis dodecanoyl-CoA + NAD+
-
 ?
1.3.1.9 trans-2-dodecenoyl-CoA + NADH + H+
-
 Mycobacterium tuberculosis ATCC 25618 dodecanoyl-CoA + NAD+
-
 ?
1.3.1.118 trans-2-dodecenoyl-CoA + NADH + H+
-
 Mycobacterium tuberculosis dodecanoyl-CoA + NAD+
-
 ?
1.3.1.118 trans-2-dodecenoyl-CoA + NADH + H+
-
 Mycobacterium tuberculosis ATCC 25618 dodecanoyl-CoA + NAD+
-
 ?

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.3.1.9 1.46
-
 trans-2-dodecenoyl-CoA pH 7.5, 25°C, mutant enzyme T266D Mycobacterium tuberculosis
1.3.1.9 2.49
-
 trans-2-dodecenoyl-CoA pH 7.5, 25°C, mutant enzyme T266E Mycobacterium tuberculosis
1.3.1.9 5.34
-
 trans-2-dodecenoyl-CoA pH 7.5, 25°C, wild-type enzyme Mycobacterium tuberculosis
1.3.1.9 7.12
-
 trans-2-dodecenoyl-CoA pH 7.5, 25°C, mutant enzyme T266A Mycobacterium tuberculosis
1.3.1.118 1.46
-
 trans-2-dodecenoyl-CoA pH 7.5, 25°C, mutant enzyme T266D Mycobacterium tuberculosis
1.3.1.118 2.49
-
 trans-2-dodecenoyl-CoA pH 7.5, 25°C, mutant enzyme T266E Mycobacterium tuberculosis
1.3.1.118 5.34
-
 trans-2-dodecenoyl-CoA pH 7.5, 25°C, wild-type enzyme Mycobacterium tuberculosis
1.3.1.118 7.12
-
 trans-2-dodecenoyl-CoA pH 7.5, 25°C, mutant enzyme T266A Mycobacterium tuberculosis

Cofactor

EC Number Cofactor Comment Organism Structure
1.3.1.9 NADH
-
 Mycobacterium tuberculosis
1.3.1.118 NADH
-
 Mycobacterium tuberculosis

General Information

EC Number General Information Comment Organism
1.3.1.9 metabolism the enzyme is an essential enzyme of the mycolic acid biosynthetic pathway Mycobacterium tuberculosis
1.3.1.118 metabolism the enzyme is an essential enzyme of the mycolic acid biosynthetic pathway Mycobacterium tuberculosis

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
1.3.1.9 74.49
-
 trans-2-dodecenoyl-CoA pH 7.5, 25°C, mutant enzyme T266D Mycobacterium tuberculosis
1.3.1.9 122.7
-
 trans-2-dodecenoyl-CoA pH 7.5, 25°C, mutant enzyme T266E Mycobacterium tuberculosis
1.3.1.9 130.6
-
 trans-2-dodecenoyl-CoA pH 7.5, 25°C, wild-type enzyme Mycobacterium tuberculosis
1.3.1.9 136.13
-
 trans-2-dodecenoyl-CoA pH 7.5, 25°C, mutant enzyme T266A Mycobacterium tuberculosis
1.3.1.118 74.49
-
 trans-2-dodecenoyl-CoA pH 7.5, 25°C, mutant enzyme T266D Mycobacterium tuberculosis
1.3.1.118 122.7
-
 trans-2-dodecenoyl-CoA pH 7.5, 25°C, mutant enzyme T266E Mycobacterium tuberculosis
1.3.1.118 130.6
-
 trans-2-dodecenoyl-CoA pH 7.5, 25°C, wild-type enzyme Mycobacterium tuberculosis
1.3.1.118 136.13
-
 trans-2-dodecenoyl-CoA pH 7.5, 25°C, mutant enzyme T266A Mycobacterium tuberculosis