Enzymatic synthesis of acrylamide: a success story not yet over

Kobayashi, Michihiko; Nagasawa, Tôru; Yamada, Hideaki

doi:10.1016/0167-7799(92)90283-2

Cited by 335 publications

(236 citation statements)

References 39 publications

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“…In both H-and L-NHase, cobalt acts as an active center for the production of acrylamide and nicotinamide. Acrylamide is manufactured at the industrial level not only in Japan but also in the United States and France (17,18).…”

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confidence: 99%

Self-subunit Swapping Chaperone Needed for the Maturation of Multimeric Metalloenzyme Nitrile Hydratase by a Subunit Exchange Mechanism Also Carries Out the Oxidation of the Metal Ligand Cysteine Residues and Insertion of Cobalt

Zhou¹,

Hashimoto²,

Kobayashi

2009

Journal of Biological Chemistry

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The incorporation of cobalt into low molecular mass nitrile hydratase (L-NHase) of Rhodococcus rhodochrous J1 has been found to depend on the ␣-subunit exchange between cobalt-free L-NHase (apo-L-NHase lacking oxidized cysteine residues) and its cobalt-containing mediator (holo-NhlAE containing Cys-SO 2 ؊ and Cys-SO ؊ metal ligands), this novel mode of post-translational maturation having been named self-subunit swapping, and NhlE having been recognized as a self-subunit swapping chaperone (Zhou, Z., Hashimoto, Y., Shiraki, K., and Kobayashi, M. (2008) Proc. Natl. Acad. Sci. U. S. A. 105, 14849 -14854). We discovered here that cobalt was inserted into both the cobaltfree NhlAE (apo-NhlAE) and the cobalt-free ␣-subunit (apo-␣-subunit) in an NhlE-dependent manner in the presence of cobalt and dithiothreitol in vitro. Matrix-assisted laser desorption ionization time-of-flight mass spectroscopy analysis revealed that the non-oxidized cysteine residues in apo-NhlAE were posttranslationally oxidized after cobalt insertion. These findings suggested that NhlE has two activities, i.e. cobalt insertion and cysteine oxidation. NhlE not only functions as a self-subunit swapping chaperone but also a metallochaperone that includes a redox function. Cobalt insertion and cysteine oxidation occurred under both aerobic and anaerobic conditions when Co 3؉ was used as a cobalt donor, suggesting that the oxygen atoms in the oxidized cysteines were derived from water molecules but not from dissolved oxygen. Additionally, we isolated apo-NhlAE after the self-subunit swapping event and found that it was recycled for cobalt transfer into L-NHase.

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Self-subunit Swapping Chaperone Needed for the Maturation of Multimeric Metalloenzyme Nitrile Hydratase by a Subunit Exchange Mechanism Also Carries Out the Oxidation of the Metal Ligand Cysteine Residues and Insertion of Cobalt

Zhou¹,

Hashimoto²,

Kobayashi

2009

Journal of Biological Chemistry

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“…This unusual active site geometry is likely to be relevant in terms of tuning the electronics of the active-site metal. With the importance of these enzymes both by virtue of their biological functions and as widely used industrial biocatalysts [29][30], there is much interest in understanding these structure-function relationships. As part of a program aimed at modeling the active site of these enzymes, we report herein the application of the DTDB methodology to the synthesis of the first octahedral Co(III) complexes of a pentadentate N 2 S 3 ligand.…”

Section: Introductionmentioning

confidence: 99%

Incorporation of thiolate donation using 2,2′-dithiodibenzaldehyde: Complexes of a pentadentate N2S3 ligand with relevance to the active site of Co nitrile hydratase

Smucker

VanStipdonk

Eichhorn

2007

Journal of Inorganic Biochemistry

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The use of 2,2′-dithiodibenzaldehyde (DTDB) as a reactant for incorporating thiolate donors into the coordination sphere of a transition metal complex without the need for protecting groups is expanded to include the synthesis of complexes with pentadentate ligands. The ligand N,N′-bis (thiosalicylideneimine)-2,2′-thiobis(ethylamine) (tsaltp) is synthesized at a cobalt center by the reaction of DTDB with a Co complex of thiobis(ethylamine). The resulting Co complexes are thus coordinated by the N 2 S 3 pentadentate ligand through two imine N atoms, two thiolate S atoms, and one thioether S atom. A dimeric, bis-thiolate-bridged complex (1) is isolated and converted to a monomeric CN adduct (2) by treatment with KCN. The N 2 S 3 coordination environment provided by the tsaltp ligand is similar to that provided by the protein donors at the active site of the nitrile hydratase enzymes, with 2 being the first octahedral Co complex reported with such a coordination sphere.

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“…In the actinomycete Rhodococcus rhodochrous J1 (8-10), which is used for the industrial production of acrylamide (11,12) and nicotinamide (13), nitrilase is strongly induced on the addition of -caprolactam to the culture medium (14). The induced nitrilase (15), which catalyzes the hydrolysis of nitrile to acid and ammonia, corresponds to 35% of all soluble protein in R. rhodochrous J1 (16).…”

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Hyper-inducible expression system for streptomycetes

Herai

Hashimoto

Higashibata

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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Streptomycetes produce useful enzymes and a wide variety of secondary metabolites with potent biological activities (e.g., antibiotics, immunosuppressors, pesticides, etc.). Despite their importance in the pharmaceutical and agrochemical fields, there have been no reports for practical expression systems in streptomycetes. Here, we developed a ''PnitA-NitR'' system for regulatory gene expression in streptomycetes based on the expression mechanism of Rhodococcus rhodochrous J1 nitrilase, which is highly induced by an inexpensive and safe inducer, -caprolactam. Heterologous protein expression experiments demonstrated that the system allowed suppressed basal expression and hyper-inducible expression, yielding target protein levels of as high as Ϸ40% of all soluble protein. Furthermore, the system functioned in important streptomycete strains. Thus, the P nitA-NitR system should be a powerful tool for improving the productivity of various useful products in streptomycetes.

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Enzymatic synthesis of acrylamide: a success story not yet over

Cited by 335 publications

References 39 publications

Self-subunit Swapping Chaperone Needed for the Maturation of Multimeric Metalloenzyme Nitrile Hydratase by a Subunit Exchange Mechanism Also Carries Out the Oxidation of the Metal Ligand Cysteine Residues and Insertion of Cobalt

Self-subunit Swapping Chaperone Needed for the Maturation of Multimeric Metalloenzyme Nitrile Hydratase by a Subunit Exchange Mechanism Also Carries Out the Oxidation of the Metal Ligand Cysteine Residues and Insertion of Cobalt

Incorporation of thiolate donation using 2,2′-dithiodibenzaldehyde: Complexes of a pentadentate N2S3 ligand with relevance to the active site of Co nitrile hydratase

Hyper-inducible expression system for streptomycetes

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