Analyzing the catalytic role of Asp97 in the methionine aminopeptidase from Escherichia coli

Cui

et al. 2014

FEMS Microbiol Lett

A self-subunit swapping chaperone is crucial for cobalt incorporation into nitrile hydratase. However, further information about its structural features is not available. The flexibility and positive charge of the C-terminal domain of the self-subunit swapping chaperone (P14K) of nitrile hydratase from Pseudomonas putida NRRL-18668 play an important role in cobalt incorporation. C-terminal domain truncation, alternation of C-terminal domain flexibility through mutant P14K(G86I), and elimination of the positive charge in the C-terminal domain sharply affected nitrile hydratase cobalt content and activity. The flexible, positively charged C-terminal domain most likely carries out an external action that allows a cobalt-free nitrile hydratase to overcome an energetic barrier, resulting in a cobalt-containing nitrile hydratase.

Section: Introductionmentioning

confidence: 99%

Effect of flexibility and positive charge of the C-terminal domain on the activator P14K function for nitrile hydratase inPseudomonas putida

Cui

et al. 2014

FEMS Microbiol Lett

“…The apoenzyme is likely to be unmodified, based on the results of previous studies on NHase [16] and a related enzyme, thiocyanate hydrolase (SCNase) [17]–[19]. Non-corrinoid cobalt has received increasing interest not only in bioinorganic chemistry but also in biotechnology, and its availability and remarkable chemical versatility make it an invaluable catalyst in the chemical industry [20]–[23].…”

Section: Introductionmentioning

confidence: 99%

Self-Subunit Swapping Occurs in Another Gene Type of Cobalt Nitrile Hydratase

et al. 2012

Self-subunit swapping is one of the post-translational maturation of the cobalt-containing nitrile hydratase (Co-NHase) family of enzymes. All of these NHases possess a gene organization of <β-subunit> <α-subunit> , which allows the activator protein to easily form a mediatory complex with the α-subunit of the NHase after translation. Here, we discovered that the incorporation of cobalt into another type of Co-NHase, with a gene organization of <α-subunit> <β-subunit> , was also dependent on self-subunit swapping. We successfully isolated a recombinant NHase activator protein (P14K) of Pseudomonas putida NRRL-18668 by adding a Strep-tag N-terminal to the P14K gene. P14K was found to form a complex [α(StrepP14K)2] with the α-subunit of the NHase. The incorporation of cobalt into the NHase of P. putida was confirmed to be dependent on the α-subunit substitution between the cobalt-containing α(StrepP14K)2 and the cobalt-free NHase. Cobalt was inserted into cobalt-free α(StrepP14K)2 but not into cobalt-free NHase, suggesting that P14K functions not only as a self-subunit swapping chaperone but also as a metallochaperone. In addition, NHase from P. putida was also expressed by a mutant gene that was designed with a <β-subunit> <α-subunit> order. Our findings expand the general features of self-subunit swapping maturation.

“…Acrylamide is manufactured at the industrial level not only in Japan but also in the United States and France , . In addition, non-corrin cobalt has been the subject of growing interest not only in bioinorganic chemistry but also in biotechnology, and its availability and remarkable chemical versatility make it an invaluable catalyst in the chemical industry − . 1 Abbreviations: NHase, nitrile hydratase; H-NHase, high-molecular mass nitrile hydratase; L-NHase, low-molecular mass nitrile hydratase; SCNase, thiocyanate hydrolase; NhhAG, H-NHase maturation mediator complex (αg 2 ); NhlAE, L-NHase maturation mediator complex (αe 2 ); pHJK19 (ΔG) , plasmid pHJK19 without the nhhG gene; H-NHase (+G) , H-NHase expressed from pHJK19; H-NHase (ΔG) , H-NHase expressed from pHJK19 (ΔG) ; R-H-NHase, reconstituted H-NHase derived from a mixture of holo-NhhAG and apo-H-NHase; DLS, dynamic light scattering; R-apo-H-NHase (NhlAE) , resultant H-NHase derived from a mixture of holo-NhlAE and apo-H-NHase; R-apo-L-NHase (NhhAG) , resultant L-NHase derived from mixture of holo-NhhAG and apo-L-NHase. …”

mentioning

confidence: 99%

“…Acrylamide is manufactured at the industrial level not only in Japan but also in the United States and France (14,15). In addition, non-corrin cobalt has been the subject of growing interest not only in bioinorganic chemistry but also in biotechnology, and its availability and remarkable chemical versatility make it an invaluable catalyst in the chemical industry (16)(17)(18)(19).…”

mentioning

confidence: 99%

Unique Biogenesis of High-Molecular Mass Multimeric Metalloenzyme Nitrile Hydratase: Intermediates and a Proposed Mechanism for Self-Subunit Swapping Maturation

et al. 2010

Rhodococcus rhodochrous J1 produces high- and low-molecular mass nitrile hydratases (H-NHase and L-NHase, respectively), depending on the inducer. The incorporation of cobalt into L-NHase has been found to depend on the α-subunit exchange between cobalt-free L-NHase (apo-L-NHase) and its cobalt-containing mediator, NhlAE (holo-NhlAE), this novel mode of post-translational maturation having been named self-subunit swapping and NhlE having been recognized as a self-subunit swapping chaperone. We discovered an H-NHase maturation mediator, NhhAG, consisting of NhhG and the α-subunit of H-NHase. The incorporation of cobalt into H-NHase was confirmed to be dependent on self-subunit swapping. For the first time, particles larger than apo-H-NHase were observed during the swapping process via dynamic light scattering measurements, suggesting the formation of intermediate complexes. On the basis of these findings, we initially proposed a possible mechanism for self-subunit swapping. Electron paramagnetic resonance analysis demonstrated that the coordination environment of a cobalt ion in holo-NhhAG is subtly different from that in H-NHase. Cobalt is inserted into cobalt-free NhhAG (apo-NhhAG) but not into apo-H-NHase, suggesting that NhhG functions not only as a self-subunit swapping chaperone but also as a metallochaperone. In addition, α-subunit swapping did not occur between apo-L-NHase and holo-NhhAG or between apo-H-NHase and holo-NhlAE in vitro. These findings revealed that self-subunit swapping is a subunit-specific reaction.