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BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES

An Aldehyde Oxidase in Developing Seeds of Arabidopsis Converts Benzaldehyde to Benzoic Acid^1,[OA]

Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, Michigan 48109–1048 (M.I., Y.-T.C., E.P.); and Department of Energy Plant Research Laboratory and Michigan Proteome Consortium, Michigan State University, East Lansing, Michigan 48824 (C.G.W.)

Arabidopsis (Arabidopsis thaliana) siliques synthesize high levels of benzoic acid (BA), which is incorporated into several glucosinolate compounds. The origin of BA in the siliques has not yet been determined. Here, we show that siliques have higher levels of benzaldehyde (BD)-oxidizing activity relative to leaves. The BD-oxidizing activity was purified from siliques in several chromatographic steps, and a 145-kD protein was identified as the enzyme most likely to possess this activity. The protein was trypsinized, and the sequence of the resulting peptides was determined by mass spectrometry, identifying it as the product of gene At1g04580, also designated as AAO4 (for ARABIDOPSIS ALDEHYDE OXIDASE4). AAO4 had previously been shown to be highly and specifically expressed in developing seeds, and its protein was shown to belong to a family of aldehyde oxidases. Here, we show that the AAO4 protein is an aldehyde oxidase that can use several substrates but that, among the substrates tested, has the lowest K_m value (23 µM) with BD. AAO4 is able to oxidize BD without NAD⁺, but its activity increases by 50% when this cofactor is added. The pH optimum of AAO4 is 7.0. Plants homozygous for a null allele in AAO4 showed a reduction of 30% to 45% in the total levels of BA in seeds as well as 7% to 9% and 32% to 38% decreases in the levels of 3-benzoyloxypropylglucosinolate and 4-benzoyloxybutylglucosinolate, respectively. Expressing AAO4 in Escherichia coli resulted in a 3-fold increase of BD-oxidizing activity in crude bacterial extracts over endogenous levels. These findings indicate that in Arabidopsis seeds, oxidation of BD contributes in part to the synthesis of BA.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Eran Pichersky (lelx{at}umich.edu).

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www.plantphysiol.org/cgi/doi/10.1104/pp.109.135848

^* Corresponding author; e-mail lelx{at}umich.edu.

Received January 18, 2009; accepted March 3, 2009; published March 18, 2009.