Abstract
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.
Highlights
Zide found in the commercial mushroom Agaricus bisporus, might have carcinogenic activity [2]
We prepared adipic acid ethylidene hydrazide (AMH) [2], which is derived from ADH when one hydrazide group remains unreacted with acetaldehyde
Because no carbon source other than AEH or VEH was included in the culture medium, these results indicated that strain MK883 metabolized the hydrazones (AEH and VEH) as both carbon and energy sources
Summary
Zide found in the commercial mushroom Agaricus bisporus, might have carcinogenic activity [2]. Hydrazide derivatives that lack substitutions on one of the nitrogen atoms (R1R2N-NH2) possess a highly active -N-NH2 moiety and react with the carbonyl groups of various compounds to produce hydrazones, which can be described as in Reaction 1. One example is the hemolytic toxin, gyromitrin (acetaldehyde methylformylhydrazone), which is produced by false morel mushrooms [4], and an alkaloid-containing hydrazone has been isolated from a marine sponge [5] Their physiological functions and biosynthetic mechanisms are unknown. The enzyme attacked the CϭN double bond of hydrazones and catalyzed NADϩ-dependent oxidation and hydration to produce the relevant hydrazide and acid. This “oxidative hydrolysis” reaction is unique among known enzymes. The present study is the first to uncover a mechanism for hydrazone assimilation in biological systems and a novel role of an enzyme in the ALDH superfamily, namely cleavage of the CϭN double bond
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