5-Cyanovaleramide production using immobilized pseudomonas chlororaphis B23

Hann, Eugenia C.; Eisenberg, Amy; Fager, Susan K.; Perkins, Neal E.; Gallagher, F. Glenn; Cooper, Susan M.; Gavagan, John E.; Stieglitz, Barry; Hennessey, Susan M.; DiCosimo, Robert

doi:10.1016/s0968-0896(99)00157-1

Cited by 95 publications

(38 citation statements)

References 22 publications

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“…These enzymes are heterodimers composed of ␣ and ␤ subunits and are classified as either ferric or cobalt nitrile hydratases, depending on the type of metal cofactor within the ␣-subunit active site (49). Nitrile hydratases have been exploited as industrial biocatalysts in the commercial synthesis of acrylamide, nicotinamide, and 5-cyanovaleramide (50,51). However, their involvement in IAA biosynthesis, specifically in the conversion of IAN into IAM, has not been well studied.…”

Section: -H-e-g-r-c-f-v-vmentioning

confidence: 99%

Characterization of a Nitrilase and a Nitrile Hydratase from Pseudomonas sp. Strain UW4 That Converts Indole-3-Acetonitrile to Indole-3-Acetic Acid

Duca

Rose

Glick

2014

Appl Environ Microbiol

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Indole-3-acetic acid (IAA) is a fundamental phytohormone with the ability to control many aspects of plant growth and development. Pseudomonas sp. strain UW4 is a rhizospheric plant growth-promoting bacterium that produces and secretes IAA. While several putative IAA biosynthetic genes have been reported in this bacterium, the pathways leading to the production of IAA in strain UW4 are unclear. Here, the presence of the indole-3-acetamide (IAM) and indole-3-acetaldoxime/indole-3-acetonitrile (IAOx/IAN) pathways of IAA biosynthesis is described, and the specific role of two of the enzymes (nitrilase and nitrile hydratase) that mediate these pathways is assessed. The genes encoding these two enzymes were expressed in Escherichia coli, and the enzymes were isolated and characterized. Substrate-feeding assays indicate that the nitrilase produces both IAM and IAA from the IAN substrate, while the nitrile hydratase only produces IAM. The two nitrile-hydrolyzing enzymes have very different temperature and pH optimums. Nitrilase prefers a temperature of 50°C and a pH of 6, while nitrile hydratase prefers 4°C and a pH of 7.5. Based on multiple sequence alignments and motif analyses, physicochemical properties and enzyme assays, it is concluded that the UW4 nitrilase has an aromatic substrate specificity. The nitrile hydratase is identified as an iron-type metalloenzyme that does not require the help of a P47K activator protein to be active. These data are interpreted in terms of a preliminary model for the biosynthesis of IAA in this bacterium.

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Section: -H-e-g-r-c-f-v-vmentioning

confidence: 99%

Characterization of a Nitrilase and a Nitrile Hydratase from Pseudomonas sp. Strain UW4 That Converts Indole-3-Acetonitrile to Indole-3-Acetic Acid

Duca

Rose

Glick

2014

Appl Environ Microbiol

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“…Two of the fruits of our application-oriented nitrile studies are the current industrial production of acrylamide and nicotinamide using the NHase of Rhodococcus rhodochrous J1 (1,9). On the other hand, the NHase of Pseudomonas chlororaphis B23 (22), which was previously used as the previous-generation catalyst for acrylamide manufacture (8,20,23), is now used for the industrial production of 5-cyanovaleramide (24). In this strain, we identified a unique hemoprotein that catalyzes the dehydration of aliphatic aldoximes to the corresponding nitriles (25), naming it aliphatic aldoxime dehydratase (OxdA); it has been approved as a unique enzyme by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB): EC 4.99.1.5 (www.chem.qmul.ac.uk/iubmb/enzyme/EC4/99/1/5.html).…”

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confidence: 99%

Crystal structure of aldoxime dehydratase and its catalytic mechanism involved in carbon-nitrogen triple-bond synthesis

Nomura

Hashimoto

Ohta

et al. 2013

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

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Aldoxime dehydratase (OxdA), which is a unique heme protein, catalyzes the dehydration of an aldoxime to a nitrile even in the presence of water in the reaction mixture. Unlike the utilization of H 2 O 2 or O 2 as a mediator of catalysis by other heme-containing enzymes (e.g., P450), OxdA is notable for the direct binding of a substrate to the heme iron. Here, we determined the crystal structure of OxdA. We then constructed OxdA mutants in which each of the polar amino acids lying within ∼6 Å of the iron atom of the heme was converted to alanine. Among the purified mutant OxdAs, S219A had completely lost and R178A exhibited a reduction in the activity. Together with this finding, the crystal structural analysis of OxdA and spectroscopic and electrostatic potential analyses of the wildtype and mutant OxdAs suggest that S219 plays a key role in the catalysis, forming a hydrogen bond with the substrate. Based on the spatial arrangement of the OxdA active site and the results of a series of mutagenesis experiments, we propose the detailed catalytic mechanism of general aldoxime dehydratases: (i) S219 stabilizes the hydroxy group of the substrate to increase its basicity; (ii) H320 acts as an acid-base catalyst; and (iii) R178 stabilizes the heme, and would donate a proton to and accept one from H320.hemoprotein | reaction mechanism | resonance Raman | distal histidine

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“…One of the fruits of our applicationoriented nitrile studies is the current industrial production of acrylamide and nicotinamide using the NHase of Rhodococcus rhodochrous J1 (1,9). On the other hand, the NHase of Pseudomonas chlororaphis B23 (22), which was previously used as a catalyst for acrylamide manufacture (8,20,23), is now used for the industrial production of 5-cyanovaleramide (24). Recently, we discovered a novel hemoprotein that catalyzes the dehydration of aliphatic aldoximes to the corresponding nitriles in P. chlororaphis B23 (25) and named it aliphatic aldoxime dehydratase (OxdA); it has been approved as a new enzyme by Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUBMB): EC 4.99.1.5.…”

mentioning

confidence: 99%

Discovery of a reaction intermediate of aliphatic aldoxime dehydratase involving heme as an active center

Konishi

Ohta²,

Oinuma³

et al. 2006

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

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5-Cyanovaleramide production using immobilized pseudomonas chlororaphis B23

Cited by 95 publications

References 22 publications

Characterization of a Nitrilase and a Nitrile Hydratase from Pseudomonas sp. Strain UW4 That Converts Indole-3-Acetonitrile to Indole-3-Acetic Acid

Characterization of a Nitrilase and a Nitrile Hydratase from Pseudomonas sp. Strain UW4 That Converts Indole-3-Acetonitrile to Indole-3-Acetic Acid

Crystal structure of aldoxime dehydratase and its catalytic mechanism involved in carbon-nitrogen triple-bond synthesis

Discovery of a reaction intermediate of aliphatic aldoxime dehydratase involving heme as an active center

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