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Literature summary for 1.3.1.9 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)

Cloned (Comment) Organism
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

Protein Variants

Protein Variants Comment Organism
T266A phosphoablative mutant with activity similar to wild-type enzyme Mycobacterium tuberculosis
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
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

KM Value [mM]

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

Organism

Organism UniProt Comment Textmining
Mycobacterium tuberculosis P9WGR1
-
-
Mycobacterium tuberculosis ATCC 25618 P9WGR1
-
-

Posttranslational Modification

Posttranslational Modification Comment Organism
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)

Purification (Comment) Organism
-
Mycobacterium tuberculosis

Substrates and Products (Substrate)

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

Turnover Number [1/s]

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

Cofactor

Cofactor Comment Organism Structure
NADH
-
Mycobacterium tuberculosis

General Information

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

kcat/KM [mM/s]

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