Low-molecular-mass nitrile hydratase from Rhodococcus rhodochrous J1: purification, substrate specificity and comparison with the analogous high-molecular-mass enzyme

Wieser, M

doi:10.1016/s0378-1097(98)00447-9

Cited by 5 publications

(9 citation statements)

References 12 publications

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“…6). This range is significantly wider than other NHases from R. rhodochrous J1 (H‐NHase, pH 6.0–8.5; Wieser et al ., 1998), P. chlororaphis B23 (pH 6.0–7.5; Nagasawa et al ., 1987) and Rhodococcus sp. N774 (pH 7.0–8.5; Endo and Watanabe, 1989), which showed the decrease of the activity at higher pH around 7.5–8.5.…”

Section: Discussionmentioning

confidence: 77%

Purification and characterization of a novel nitrile hydratase from Rhodococcus sp. RHA1

Okamoto

Eltis²

2007

Molecular Microbiology

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Section: Discussionmentioning

confidence: 77%

Purification and characterization of a novel nitrile hydratase from Rhodococcus sp. RHA1

Okamoto

Eltis²

2007

Molecular Microbiology

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“…The kinetic parameters of P. thermophila NHase, with regard to the size of substrates, were similar to those of the low‐molecular‐mass Co‐type NHase from R. rhodochrous J1 [13]. Three putative residues that determine the substrate specificity of P. thermophila NHase, i.e.…”

Section: Substrate Specificity Comparison With Other Nhasesmentioning

confidence: 90%

Mutational and structural analysis of cobalt‐containing nitrile hydratase on substrate and metal binding

Miyanaga

Fushinobu

Itō

et al. 2003

European Journal of Biochemistry

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Mutants of a cobalt-containing nitrile hydratase (NHase, EC 4.2.1.84) from Pseudonocardia thermophila JCM 3095 involved in substrate binding, catalysis and formation of the active center were constructed, and their characteristics and crystal structures were investigated. As expected from the structure of the substrate binding pocket, the wild-type enzyme showed significantly lower K m and K i values for aromatic substrates and inhibitors, respectively, than aliphatic ones. In the crystal structure of a complex with an inhibitor (n-butyric acid) the hydroxyl group of bTyr68 formed hydrogen bonds with both n-butyric acid and aSer112, which is located in the active center. The bY68F mutant showed an elevated K m value and a significantly decreased k cat value. The apoenzyme, which contains no detectable cobalt atom, was prepared from Escherichia coli cells grown in medium without cobalt ions. It showed no detectable activity. A disulfide bond between aCys108 and aCys113 was formed in the apoenzyme structure. In the highly conserved sequence motif in the cysteine cluster region, two positions are exclusively conserved in cobalt-containing or iron-containing nitrile hydratases. Two mutants (aT109S and aY114T) were constructed, each residue being replaced with an iron-containing one. The aT109S mutant showed similar characteristics to the wild-type enzyme. However, the aY114T mutant showed a very low cobalt content and catalytic activity compared with the wild-type enzyme, and oxidative modifications of aCys111 and aCys113 residues were not observed. The aTyr114 residue may be involved in the interaction with the nitrile hydratase activator protein of P. thermophila.

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“…The NHase within the resting cells of R. rhodochrous PA-34 showed wide optimum temperature from 30°C to 60°C, which further indicate that this enzyme was more stable within the cells. respectively [15].…”

Section: Effect Of Temperature On Activity and Stability Of Nhasementioning

confidence: 99%

“…Below and above 40°C, NHase activity of R. rhodochrous PA-34 decreased drastically. Generally, the mesophilic NHases have exhibited maximum activity near ambient temperature between 20 and 40°C [14,15]. However, thermophilic NHases of Bacillus RAPc8 [16] and Pseudonocardia thermophila [17] exhibited maximum activity at 60°C.…”

Section: Effect Of Temperature On Activity and Stability Of Nhasementioning

confidence: 99%

Purification of a hyperactive nitrile hydratase from resting cells of Rhodococcus rhodochrous PA-34

2009

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A propionitrile-induced nitrile hydratase (NHase), a promising biocatalyst for synthesis of organic amides has been purifi ed from cell-free extract of Rhodococcus rhodochrous PA-34. About 11-fold purifi cation of NHase was achieved with 52% yield. The SDS-PAGE of the purifi ed enzyme revealed that it consisted of two subunits of 25.04 kD and 30.6 kD. However, the molecular weight of holoenzyme was speculated to be 86 kD by native-PAGE. This NHase exhibited maximum activity at pH 8.0 and temperature 40°C. Half-life was 2 h at 40°C and 0.5 h at 50°C. The Km and Vmax were 167 mM and 250 μmole/min/mg using 25 mM 3-cyanopyridine as substrate. AgNO 3 , Pb(CH 3 COO) 2 and HgCl 2 inhibited the NHase to extent of 89-100%.

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Low-molecular-mass nitrile hydratase from Rhodococcus rhodochrous J1: purification, substrate specificity and comparison with the analogous high-molecular-mass enzyme

Cited by 5 publications

References 12 publications

Purification and characterization of a novel nitrile hydratase from Rhodococcus sp. RHA1

Purification and characterization of a novel nitrile hydratase from Rhodococcus sp. RHA1

Mutational and structural analysis of cobalt‐containing nitrile hydratase on substrate and metal binding

Purification of a hyperactive nitrile hydratase from resting cells of Rhodococcus rhodochrous PA-34

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