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Literature summary for 1.14.13.69 extracted from

  • Sims, L.P.; Lockwood, C.W.J.; Crombie, A.T.; Bradley, J.M.; Le Brun, N.E.; Murrell, J.C.
    Purification and characterization of the isoprene monooxygenase from Rhodococcus sp. strain AD45 (2022), Appl. Environ. Microbiol., 88, e0002922 .
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

Cloned (Comment) Organism
the oxygenase component of IsoMO from Rhodococcus sp. strain AD45 is recombinantly expressed as functional protein in Rhodococcus sp. strain AD45-ID from plasmid pTipQC2S2:isoEx, which encodes the complete IsoMO gene cluster, including an N-terminal StrepII tag on IsoA. Recombinant expression of N-terminally His6-tagged IsoC in Escherichia coli. Recombinant expression of N-terminally StrepII-tagged IsoD in Escherichia coli strain Rosetta2/(pLysS) using plasmid pET51b:isoD. Expression of His-tagged, untagged, glutathione S-transferase (GST)-fusion and maltose binding protein (MBP)-fusion IsoF, via both homologous expression in Rhodococcus sp. AD45-ID and heterologous expression in Escherichia coli Rosetta2/(pLysS), results in protein which is either expressed entirely in inclusion bodies, expressed so poorly it is impossible to purify, or when purified does not contain the predicted cofactors or demonstrate the ability to reduce IsoC Rhodococcus sp. AD45

Inhibitors

Inhibitors Comment Organism Structure
propyne
-
Rhodococcus sp. AD45

Metals/Ions

Metals/Ions Comment Organism Structure
Fe ICP-MS analysis of purified IsoABE is used to confirm the presence of the predicted diiron center, presence of 1.12 Fe per monomer, which is lower than the predicted 2 Fe/monomer. Purified IsoMO oxygenase is thus deficient in Fe cofactor and is expected to exhibit decreased activity as a result Rhodococcus sp. AD45

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
isoprene + NADH + H+ + O2 Rhodococcus sp. AD45
-
isoprene epoxide + NAD+ + H2O
-
?

Organism

Organism UniProt Comment Textmining
Rhodococcus sp. AD45
-
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant StrepII-tagged oxygenase component IsoABE from Rhodococcus sp. strain AD45 and recombinant N-terminally His6-tagged IsoC from Escherichia coli both by affinity chromatography and gel filtration, the latter to over 90% purity. Recombinant StrepII-tagged IsoD from Escherichia coli strain Rosetta2 by affinity, anion exchange, and hydrophobic interaction chromatography, and gel filtration, the N-terminal methionine of IsoD has been removed Rhodococcus sp. AD45

Renatured (Commentary)

Renatured (Comment) Organism
reconstitution of the active isoprene monooxygenase complex from Rhodococcus sp. strain AD45 in the presence of Rhodococcus sp. strain AD45 cell lysate Rhodococcus sp. AD45

Source Tissue

Source Tissue Comment Organism Textmining
culture condition:isoprene-grown cell
-
Rhodococcus sp. AD45
-

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
0.0041
-
purified recombinant oxygenase component IsoABE, pH 7.0, temperature not specified in the publication Rhodococcus sp. AD45

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
isoprene + NADH + H+ + O2
-
Rhodococcus sp. AD45 isoprene epoxide + NAD+ + H2O
-
?
additional information product identification after reaction of th recombinant enzyme with isoprene by GC-MS Rhodococcus sp. AD45 ?
-
-

Subunits

Subunits Comment Organism
More the four component IsoMO is likely to comprise an oxygenase (IsoABE), reductase (IsoF), coupling protein (IsoD) and Rieske-type ferredoxin (IsoC). The alpha subunit of the oxygenase component of SDIMOs (equivalent to IsoA in IsoMO) contains an active site diiron center required for catalytic activity of the enzyme. The oxygenase component might form dimers in an (alphabetagamma)2 arrangement. The beta subunit (IsoE) and the gamma subunit (IsoB) combine with the alpha subunit to form the oxygenase complex Rhodococcus sp. AD45

Synonyms

Synonyms Comment Organism
IsoMO
-
Rhodococcus sp. AD45
isoprene monooxygenase
-
Rhodococcus sp. AD45

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7
-
assay at Rhodococcus sp. AD45

Cofactor

Cofactor Comment Organism Structure
FAD
-
Rhodococcus sp. AD45
Ferredoxin only contains a Rieske-type [2Fe-2S] cluster, characterization of the Rieske protein component (IsoC) of isoprene monooxygenase from Rhodococcus sp. AD45, overview Rhodococcus sp. AD45
additional information the reductase component contains two cofactors, an FAD and a [2Fe-2S] cluster, whereas the ferredoxin only contains a Rieske-type [2Fe-2S] cluster. These redox active components are involved in the transfer of electrons, from NADH via the reductase, and the Rieske protein in four-component SDIMOs, to the diiron active site of the oxygenase Rhodococcus sp. AD45
NADH
-
Rhodococcus sp. AD45
[2Fe-2S]-center
-
Rhodococcus sp. AD45

General Information

General Information Comment Organism
evolution isoprene monooxygenase (IsoMO) is a soluble diiron center monooxygenase in the same family of oxygenases as soluble methane monooxygenase, alkene monooxygenase, and toluene monooxygenase Rhodococcus sp. AD45
additional information the endogenous IsoF component may be required to restore activity Rhodococcus sp. AD45