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

Konishi, Kazuhiko; Ohta, Takao; Oinuma, Ken-Ichi; Hashimoto, Yoshiteru; Kitagawa, Teizo; Kobayashi, Michihiko

doi:10.1073/pnas.0505412103

Cited by 31 publications

(41 citation statements)

References 55 publications

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“…observed on mixing of ferrous H320A with butyraldoxime (43), and it has the structure Fe(II)Por-N=CH-R. On the other hand, the reaction intermediate (named OS-II) after the electrophilic attack by H320 on the substrate is observed on mixing of the ferrous wild-type with a large amount of butyraldoxime (final 200 mM) under ferrous condition (33). OS-II may be in the resonance structures of Fe(III)Por-N=CH-R and Fe(IV) Por=N − =CH-R.…”

Section: Discussionmentioning

confidence: 99%

“…Therefore, limitation of the substrate mobility by S219 is likely to be required for the start of a catalytic reaction. We have already discovered two types of OxdA-substrate complexes (33), and here at first predicted that S219 is involved in the further processing of either or both of these complexes. The two reaction intermediates are observed before and after electrophilic attack on the substrate by H320, respectively.…”

Section: Discussionmentioning

confidence: 99%

“…We previously reported that ferrous OxdA, containing a five-coordinated high-spin heme and H299 as its proximal ligand, is the reactive form of the enzyme (31,32), and that H320 in the distal heme pocket plays a crucial role in donating a proton to the substrate during aldoxime dehydration (32). This enzyme is not only academically interesting (25,(32)(33)(34)(35)(36)(37)(38), but is also expected to be applicable to the practical production of nitriles (39) because the enzyme reaction occurs under mild conditions, in contrast to the chemical dehydration of aldoximes under harsh conditions. Unless the detailed reaction mechanism is clearly understood, it would be difficult to enhance the enzyme activity efficiently for nitrile synthesis.…”

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confidence: 99%

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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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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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confidence: 99%

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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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“…However, information on the metabolism of an isonitrile at the protein and gene levels had been quite limited until we discovered an isonitrile-degrading enzyme. We have extensively studied the biological metabolism of isonitriles and revealed that it is quite different from nitrile metabolism (20)(21)(22)(23). We initially found isonitrile hydratase, which catalyzes the hydration of an isonitrile (R-N'C) to the corresponding N-substituted formamide [R-NH-CH(AO)] (24,25).…”

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confidence: 99%

A New Synthetic Route to N -Benzyl Carboxamides through the Reverse Reaction of N -Substituted Formamide Deformylase

Hashimoto

Sakashita

Fukatsu

et al. 2014

Appl Environ Microbiol

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Here, we discovered that this enzyme catalyzed the reverse reaction, synthesizing N-benzylformamide (NBFA) from benzylamine and formate. The reverse reaction proceeded only in the presence of high substrate concentrations. The effects of pH and inhibitors on the reverse reaction were almost the same as those on the forward reaction, suggesting that the forward and reverse reactions are both catalyzed at the same catalytic site. Bisubstrate kinetic analysis using formate and benzylamine and dead-end inhibition studies using a benzylamine analogue, aniline, revealed that the reverse reaction of this enzyme proceeds via an ordered two-substrate, two-product (bi-bi) mechanism in which formate binds first to the enzyme active site, followed by benzylamine binding and the subsequent release of NBFA. To our knowledge, this is the first report of the reverse reaction of an amine-forming deformylase. Surprisingly, analysis of the substrate specificity for acids demonstrated that not only formate, but also acetate and propionate (namely, acids with numbers of carbon atoms ranging from C 1 to C 3 ), were active as acid substrates for the reverse reaction. Through this reaction, N-substituted carboxamides, such as NBFA, N-benzylacetamide, and N-benzylpropionamide, were synthesized from benzylamine and the corresponding acid substrates.

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“…We have extensively studied the biological metabolism of toxic compounds containing a CϵN moiety, such as nitriles (1)(2)(3)(4)(5) and isonitriles (6 -9). Isonitrile (more generally called isocyanide) containing an isocyano group (ϪNϵC) is an isomer of nitrile.…”

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Novel Isonitrile Hydratase Involved in Isonitrile Metabolism

Sato¹,

Hashimoto²,

Fukatsu

et al. 2010

Journal of Biological Chemistry

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We previously discovered N-substituted formamide deformylase (NfdA) in Arthrobacter pascens F164, which degrades N-substituted formamide (Fukatsu, H., Hashimoto, Y., Goda, M., Higashibata, H., and Kobayashi, M. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 13726 -13731). In this study, we found an enzyme involved in the first step of isonitrile metabolism, isonitrile hydratase, that hydrates isonitrile to the corresponding N-substituted formamide. First, we investigated the optimum culture conditions for the production of isonitrile hydratase. The highest enzyme activity was obtained when A. pascens F164 was cultured in a nutrient medium containing N-benzylformamide. This Arthrobacter isonitrile hydratase was purified, characterized, and compared with Pseudomonas putida N19-2 isonitrile hydratase (InhA), which is the sole one reported at present. Arthrobacter isonitrile hydratase was found to have a molecular mass of about 530 kDa and to consist of 12 identical subunits. The apparent K m value for cyclohexyl isocyanide was 0.95 ؎ 0.05 mM. A. pascens F164 grew and exhibited the isonitrile hydratase and N-substituted formamide deformylase activities when cultured in a medium containing an isonitrile as the sole carbon and nitrogen sources. However, both enzyme activities were not observed on culture in a medium containing glycerol and (NH 4 ) 2 SO 4 as the sole carbon and nitrogen sources, respectively. These findings suggested that the Arthrobacter enzyme is an inducible enzyme, possibly involved in assimilation and/or detoxification of isonitrile. Moreover, gene cloning of the Arthrobacter enzyme revealed no sequence similarity between this enzyme and InhA. Comparison of their properties and features demonstrated that the two enzymes are biochemically, immunologically, and structurally different from each other. Thus, we discovered a new isonitrile hydratase named InhB.We have extensively studied the biological metabolism of toxic compounds containing a CϵN moiety, such as nitriles (1-5) and isonitriles (6 -9). Isonitrile (more generally called isocyanide) containing an isocyano group (ϪNϵC) is an isomer of nitrile. Like nitrile, isonitrile is generally highly toxic and produced in nature, probably for self-defense, by various organisms, including bacteria, fungi, marine sponges, etc. (10, 11). For example, xanthocillin was first isolated as an isocyano metabolite from Penicillium notatum, and this isonitrile exhibits a wide antibiotic activity spectrum (12). There have been many other reports about such naturally occurring isocyanides, but information is quite limited on the metabolism of isonitrile. Some metabolic isonitrile intermediates have been elucidated through incorporation experiments, and we initially reported two enzymes involved in isonitrile metabolism. First, we discovered isonitrile hydratase in Pseudomonas putida N19-2, which catalyzes the hydration of an isonitrile to the corresponding N-substituted formamide (R-NH-CHO) (6, 7). It has been approved as a new enzyme by the Nomenclature Committee of t...

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

Cited by 31 publications

References 55 publications

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

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

A New Synthetic Route to N -Benzyl Carboxamides through the Reverse Reaction of N -Substituted Formamide Deformylase

Novel Isonitrile Hydratase Involved in Isonitrile Metabolism

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