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J. Biol. Chem., Vol. 283, Issue 9, 5790-5800, February 29, 2008

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Novel Dehydrogenase Catalyzes Oxidative Hydrolysis of Carbon-Nitrogen Double Bonds for Hydrazone Degradation^*

From the Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan

Hydrazines and their derivatives are versatile artificial and natural compounds that are metabolized by elusive biological systems. Here we identified microorganisms that assimilate hydrazones and isolated the yeast, Candida palmioleophila MK883. When cultured with adipic acid bis(ethylidene hydrazide) as the sole source of carbon, C. palmioleophila MK883 degraded hydrazones and accumulated adipic acid dihydrazide. Cytosolic NAD⁺- or NADP⁺-dependent hydrazone dehydrogenase (Hdh) activity was detectable under these conditions. The production of Hdh was inducible by adipic acid bis(ethylidene hydrazide) and the hydrazone, varelic acid ethylidene hydrazide, under the control of carbon catabolite repression. Purified Hdh oxidized and hydrated the C=N double bond of acetaldehyde hydrazones by reducing NAD⁺ or NADP⁺ to produce relevant hydrazides and acetate, the latter of which the yeast assimilated. The deduced amino acid sequence revealed that Hdh belongs to the aldehyde dehydrogenase (Aldh) superfamily. Kinetic and mutagenesis studies showed that Hdh formed a ternary complex with the substrates and that conserved Cys is essential for the activity. The mechanism of Hdh is similar to that of Aldh, except that it catalyzed oxidative hydrolysis of hydrazones that requires adding a water molecule to the reaction catalyzed by conventional Aldh. Surprisingly, both Hdh and Aldh from baker's yeast (Ald4p) catalyzed the Hdh reaction as well as aldehyde oxidation. Our findings are unique in that we discovered a biological mechanism for hydrazone utilization and a novel function of proteins in the Aldh family that act on C=N compounds.

Received for publication, November 2, 2007 , and in revised form, December 17, 2007.

The nucleotide sequence(s) reported in this paper has been submitted to the DDBJ/GenBank^TM/EBI Data Bank with accession number(s) AB361432, AB361594, AB361592, AB361593, AB361588, AB361589, AB361590, and AB361591.

^* This work was partly supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Culture, and Sports of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental text, references, Figs. S1 and S2, and Tables S1 and S2.

¹ To whom correspondence should be addressed. Tel./Fax: 81-298-53-4937; E-mail: ntakaya{at}sakura.cc.tsukuba.ac.jp.

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