Functional expression of nitrile hydratase in Escherichia coli: requirement of a nitrile hydratase activator and post-translational modification of a ligand cysteine.
The nitrile hydratase (NHase) from Rhodococcus sp. N-771 is a photoreactive enzyme that is inactivated on nitrosylation of the non-heme iron center and activated on photo-dissociation of nitric oxide (NO). The nitrile hydratase operon consists of six genes encoding NHase regulator 2, NHase regulator 1, amidase, NHase alpha subunit, NHase beta subunit and NHase activator. We overproduced the NHase in Escherichia coli using a T7 expression system. The NHase was functionally expressed in E. coli only when the NHase activator encoded downstream of the beta subunit gene was co-expressed and the transformant was grown at 30 degrees C or less. A ligand cysteine, alphaCys112, of the recombinant NHase was also post-translationally modified to a cysteine-sulfinic acid similar to for the native NHase. Although another modification of alphaCys114 could not be identified because of the instability under acidic conditions, the recombinant NHase could be reversibly inactivated by nitric oxide.
Nojiri
M
M
The Institute of Physical and Chemical Research (RIKEN), Wako, Saitama, 351-0198, Japan.
Yohda
M
M
Odaka
M
M
Matsushita
Y
Y
Tsujimura
M
M
Yoshida
T
T
Dohmae
N
N
Takio
K
K
Endo
I
I
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003545:Cysteine / Q000737:chemistry; D017931:DNA Primers / Q000235:genetics; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D015870:Gene Expression; D005798:Genes, Bacterial; D006836:Hydro-Lyases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008024:Ligands; D008969:Molecular Sequence Data; D009876:Operon; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012240:Rhodococcus / Q000201:enzymology / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D012995:Solubility; D013696:Temperature
D017931:DNA Primers; D008024:Ligands; D011994:Recombinant Proteins; D006836:Hydro-Lyases; C048831:nitrile hydratase; D003545:Cysteine