37[Noda Institute for Scientific Research GRANT]2006 Young Investigator Research Grant
Outline of Research Result

Analysis of Reaction Mechanism of Aldoxime Dehydrataseand Its Application to Production of Useful Compounds
Yoshiteru HASHIMOTO
Graduate School of Life and Environmental Sciences, University of Tsukuba

Research aims
We have extensively studied the biological metabolism oftoxic compounds (that have a triple bond between carbonand nitrogen) such as nitriles [R–CN] and isonitriles[R–NC]. Recently, we discovered a novel hemoproteinthat catalyzes the dehydration of aliphatic aldoximes to thecorresponding nitriles in Pseudomonas chlororaphis B23,
which was previously used as a catalyst for acrylamide
manufacture, and is now used for the industrial productionof 5-cyanovaleramide. We named it aliphatic aldoxime dehydratase (OxdA); it has been approved as a new enzyme
by NC-IUBMB: EC 4.99.1.5. (http://www.chem.qmul.ac.
uk/iubmb/enzyme/EC4/99/1/5.html). The OxdA reaction isvery unique and intriguing in the following: (i) dehydrationof the substrate in an aqueous solution; (ii) utilization of thefavorable hydrophobic environment of the heme pocket on
catalysis of dehydration; and (iii) synthesis of a C–N triplebond, which is a highly toxic functional group. The OxdA
reaction is not only academically interesting but also expected to be applicable to the practical production of nitriles because it occurs under mild conditions, in contrast
with the chemical dehydration of aldoximes under harshconditions. The reaction mechanism of OxdA has yet to beexplored, because reaction intermediates in the catalytic
cycle are not understood well. We previously reported thatferrous OxdA, containing a 5-coordinated high spin heme
and His299 as its proximal ligand, is the reactive form of
the enzyme, and that His320 in the distal heme pocket
would play a crucial role in donating a proton to a substrateduring aldoxime dehydration. We recently detected an initial reaction intermediate (named ‘OS-I’), the heme coordination structure of which was suggestive of Fe(II)–

N(OH)CH–R, butyraldoxime being used as the substrate.
However, information on the reaction intermediate of OxdA
has been quite limited and, thus, the reaction mechanism ofOxdA is not yet understood well.
Methods
Ferrous OxdA, which is an active form of OxdA, wasprepared by adding sodium dithionite to purified OxdA
under anaerobic conditions. The reaction product (butyronitrile) was determined with a gas chromatograph (GC14BPF; Shimadzu, Kyoto, Japan) equipped with a flame
ionization detector and a glass column packed with
Gaskuropack 56 (80/100% mesh; GL-Science, Tokyo,Japan).
The absorption spectrum was recorded with a Shimadzu
UV-1700 PharmaSpec spectrophotometer (Kyoto, Japan).
Resonance Raman spectra were obtained with excitation at413.1 nm with a Kr ion laser (Spectra Physics, model2060).
Results
We found that the addition of a large amount of butyraldoxime to a solution of ferrous OxdA with a low enzyme
concentration yields a novel long-lived OxdA-substratecomplex (named ‘OS-II’), whose UV-vis spectrum exhibitsa Soret peak at 415.5 nm. A gradual decrease in the Soretpeak of OS-II and a concomitant increase in the Soret peakof ferrous OxdA occurred 10 min after the addition of butyraldoxime, the spectrum of ferrous OxdA finally beingobserved. Although the reaction rate on synthesis of butyronitrile was reduced, the stoichiometric conversion of bu-38

tyraldoxime to butyronitrile was confirmed by gas chromatography. In order to obtain a more detailed insight into
OS-II, we have carried out resonance Raman (RR) spectroscopic studies on OS-II. Intriguingly, the frequency of then 4 band at 1379 cm1 of OS-II was even higher than that offerric OxdA at 1376 cm1. OS-II would be considered tocontain a highly oxidized heme compared with that involved in ferric OxdA. We used [15N]butyraldoxime(C3H7CH15NOH), and observed the butyraldoxime-isotope dependence of the RR spectra of OS-II. Intriguingly,
the 857 cm1 vibrational frequency is much higher than theFe–N single bond stretching frequency (500–600 cm1) observed for the NO adducts of hemoproteins. These findings
suggest that the bond order between the heme iron and thenitrogen atom of the substrate part in OS-II is more thanone; an FeN double bond may be formed. We propose apossible mechanism for the creation of a carbon-nitrogen
triple bond from aldoxime by OxdA.
Conclusion
Herein, we demonstrated for the first time that aldoxime
dehydratase, a unique heme-containing enzyme, forms anovel reaction intermediate with a highly oxidized heme bydirectly binding a substrate with its heme iron.
References
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Oinuma, K-I., Hashimoto, Y., Konishi, K., Goda, M., Noguchi, T.,
Higashibata, H. & Kobayashi, M. “Novel aldoxime dehydratase involved in carbon-nitrogen triple bond synthesis of Pseudomonas
chlororaphis B23: Sequencing, gene expression, purification andcharacterization” J. Biol. Chem., 278, 29600–29608 (2003)
Oinuma, K-I., Ohta, T., Konishi, K., Hashimoto, Y., Higashibata, H.,
Kitagawa, T., & Kobayashi, M. “Heme environment in aldoxime dehydratase involved in carbon-nitrogen triple bond synthesis” FEBS
Lett., 568, 44–48 (2004)
Konishi, K., Ishida, K., Oinuma, K-I., Ohta, T., Hashimoto, Y., Higashibata, H., Kitagawa, T., & Kobayashi, M. “Identification of crucial histidines involved in carbon-nitrogen triple bond synthesis byaldoxime dehydratase” J. Biol. Chem., 279, 47619–47625 (2004)
Oinuma, K-I., Kumita, H., Ohta, T., Konishi, K., Hashimoto, Y., Higashibata, H., Kitagawa, T., Shiro, Y. & Kobayashi, M. “Stoppedflow spectrophotometric and resonance Raman analyses of aldoxime
dehydratase involved in carbon-nitrogen triple bond synthesis” FEBS
Lett., 579, 1394–1398 (2005)
Konishi, K., Ohta, T., Oinuma, K-I., Hashimoto, Y., Kitagawa, T. &Kobayashi, M. “Discovery of a reaction intermediate of aliphatic aldoxime dehydratase involving heme as an active center” Proc. Natl.
Acad. Sci. USA, 103, 564–568 (2006)