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Literature summary extracted from

  • Okamoto, S.; Eltis, L.D.
    Purification and characterization of a novel nitrile hydratase from Rhodococcus sp. RHA1 (2007), Mol. Microbiol., 65, 828-838.
    View publication on PubMed

Application

EC Number Application Comment Organism
4.2.1.84 environmental protection degradation of nitrile waste Rhodococcus sp.

Inhibitors

EC Number Inhibitors Comment Organism Structure
4.2.1.84 acetamide 10 mM, product inhibition, reduction of acrylonitrile degrading activity from 1.2 micromol/min/mg to 1.0 micromol/min/mg Rhodococcus sp.
4.2.1.84 CuCl2 1 mM, strong inhibition of EDTA-dialysed enzyme, 19% remaining activity Rhodococcus sp.
4.2.1.84 ethanolamine markedly lower activity Rhodococcus sp.
4.2.1.84 Fe2+ 1 mM, inhibition of EDTA-dialysed enzyme, 38% remaining activity Rhodococcus sp.
4.2.1.84 propioamide 10 mM, product inhibition, reduction of acrylonitrile degrading activity from 1.2 micromol/min/mg to 0.91 micromol/min/mg Rhodococcus sp.
4.2.1.84 Zn2+ 1 mM, inhibition of EDTA-dialysed enzyme, 56% remaining activity Rhodococcus sp.

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
4.2.1.84 0.38
-
Propionitrile +/- 0.1, 10 mM HEPES, pH 7.5, 37°C Rhodococcus sp.
4.2.1.84 1.9
-
Butyronitrile +/- 0.2, 10 mM HEPES, pH 7.5, 37°C Rhodococcus sp.
4.2.1.84 5.4
-
acetonitrile +/- 0.3, 10 mM HEPES, pH 7.5, 37°C, Michaelis-Menten behavior at acetonitrile concentrations from 0.1 mM to 50 mM Rhodococcus sp.
4.2.1.84 6.4
-
acrylonitrile +/- 0.3, 10 mM HEPES, pH 7.5, 37°C Rhodococcus sp.

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
4.2.1.84 Co2+ component of enzyme, one ion per holoenzyme, ICP-MS Rhodococcus sp.
4.2.1.84 Co2+ increase of activity of EDTA-dialysed enzyme, 120% activity Rhodococcus sp.
4.2.1.84 Cu2+ component of enzyme, two ions per holoenzyme, ICP-MS Rhodococcus sp.
4.2.1.84 Mg2+ increase of activity of EDTA-dialysed enzyme, 130% activity Rhodococcus sp.
4.2.1.84 additional information metal content is unusual for NHases Rhodococcus sp.
4.2.1.84 Zn2+ component of enzyme, one ion per holoenzyme, ICP-MS Rhodococcus sp.

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
4.2.1.84 55746
-
alpha,beta2, 1 * 55746 + 2 * 63001, 32% amino acid sequence identity of subunits, MALDI-TOF MS and HPLC Rhodococcus sp.
4.2.1.84 63001
-
alpha,beta2, 1 * 55746 + 2 * 63001, 32% amino acid sequence identity of subunits, MALDI-TOF MS and HPLC Rhodococcus sp.
4.2.1.84 364000
-
proposal for holoenzyme subunit composition: alpha2 beta4, based on calibrated gel filtration and absorption spectra Rhodococcus sp.

Organism

EC Number Organism UniProt Comment Textmining
4.2.1.84 Rhodococcus sp.
-
-
-
4.2.1.84 Rhodococcus sp. RHA1
-
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
4.2.1.84 anion exchange and hydrophobic interaction chromatography, yield of purified protein: 1.9% (specific activity 5.9 U/mg), cytosolic proteom of cells grown on 0.1 M acetonitrile and acetic acid/ammonia are compared via quantitative 2D gel electrophoresis, spot identification by MS, no significant amino acid sequence similarity with other NHases Rhodococcus sp.

Specific Activity [micromol/min/mg]

EC Number Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
4.2.1.84 2.6
-
+/- 0.2, acrylonitrile, 50 mM, coupled assay with amidase Rhodococcus sp.
4.2.1.84 2.8
-
+/- 0.2, acrylonitrile, 50 mM, HPLC-based assay Rhodococcus sp.
4.2.1.84 5.7
-
+/- 0.2, acetonitrile, 50 mM, HPLC-based assay Rhodococcus sp.
4.2.1.84 5.9
-
+/- 0.4, acetonitrile, 50 mM, coupled assay with amidase Rhodococcus sp.

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
4.2.1.84 acetonitrile + H2O substrate specificity: acetonitrile ~ propionitrile > acrylonitrile >> butyronitrile Rhodococcus sp. acetamide
-
r
4.2.1.84 acetonitrile + H2O substrate specificity: acetonitrile ~ propionitrile > acrylonitrile >> butyronitrile Rhodococcus sp. RHA1 acetamide
-
r
4.2.1.84 acrylonitrile + H2O substrate specificity: acetonitrile ~ propionitrile > acrylonitrile >> butyronitrile Rhodococcus sp. acrylamide
-
r
4.2.1.84 acrylonitrile + H2O substrate specificity: acetonitrile ~ propionitrile > acrylonitrile >> butyronitrile Rhodococcus sp. RHA1 acrylamide
-
r
4.2.1.84 butyronitrile + H2O substrate specificity: acetonitrile ~ propionitrile > acrylonitrile >> butyronitrile Rhodococcus sp. butyramide
-
r
4.2.1.84 butyronitrile + H2O substrate specificity: acetonitrile ~ propionitrile > acrylonitrile >> butyronitrile Rhodococcus sp. RHA1 butyramide
-
r
4.2.1.84 additional information no detectably transformation with 2-methyl-2-butenenitrile, benzonitrile and phenylacetonitrile Rhodococcus sp. ?
-
?
4.2.1.84 additional information no detectably transformation with 2-methyl-2-butenenitrile, benzonitrile and phenylacetonitrile Rhodococcus sp. RHA1 ?
-
?
4.2.1.84 propionitrile + H2O substrate specificity: acetonitrile ~ propionitrile > acrylonitrile >> butyronitrile Rhodococcus sp. propionamide
-
r
4.2.1.84 propionitrile + H2O substrate specificity: acetonitrile ~ propionitrile > acrylonitrile >> butyronitrile Rhodococcus sp. RHA1 propionamide
-
r

Subunits

EC Number Subunits Comment Organism
4.2.1.84 heterotrimer alpha,beta2, 1 * 55746 + 2 * 63001, 32% amino acid sequence identity of subunits, MALDI-TOF MS and HPLC Rhodococcus sp.
4.2.1.84 More sequence similarity and size of subunits is unusual for NHases Rhodococcus sp.

Synonyms

EC Number Synonyms Comment Organism
4.2.1.84 ANHase acetonitrile hydratase Rhodococcus sp.
4.2.1.84 NHase
-
Rhodococcus sp.
4.2.1.84 nitrile hydratase
-
Rhodococcus sp.

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
4.2.1.84 0.99
-
Butyronitrile +/- 0.04, 10 mM HEPES, pH 7.5, 37°C Rhodococcus sp.
4.2.1.84 2 8 acrylonitrile +/- 0.5, 10 mM HEPES, pH 7.5, 37°C Rhodococcus sp.
4.2.1.84 2.6
-
Propionitrile +/- 0.2, 10 mM HEPES, pH 7.5, 37°C Rhodococcus sp.
4.2.1.84 41
-
acetonitrile +/- 0.9, 10 mM HEPES, pH 7.5, 37°C Rhodococcus sp.

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
4.2.1.84 9
-
-
Rhodococcus sp.

pH Range

EC Number pH Minimum pH Maximum Comment Organism
4.2.1.84 5.5 10 activity drops sharply below pH 5.5 and above pH 10.0 Rhodococcus sp.