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J. Biol. Chem., Vol. 279, Issue 17, 16947-16953, April 23, 2004

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Characterization and Metabolic Function of a Peroxisomal Sarcosine and Pipecolate Oxidase from Arabidopsis^*

Aymeric Goyer, Tanya L. Johnson, Laura J. Olsen, Eva Collakova¶, Yair Shachar-Hill¶, David Rhodes||, and Andrew D. Hanson**

From the Horticultural Sciences Department, University of Florida, Gainesville, Florida 32611, the Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, Michigan 48109, the ^¶Department of Plant Biology, Michigan State University, East Lansing, Michigan 48824, and the ^||Department of Horticulture, Purdue University, West Lafayette, Indiana 47907

Sarcosine oxidase (SOX) is known as a peroxisomal enzyme in mammals and as a sarcosine-inducible enzyme in soil bacteria. Its presence in plants was unsuspected until the Arabidopsis genome was found to encode a protein (AtSOX) with 33% sequence identity to mammalian and bacterial SOXs. When overexpressed in Escherichia coli, AtSOX enhanced growth on sarcosine as sole nitrogen source, showing that it has SOX activity in vivo, and the recombinant protein catalyzed the oxidation of sarcosine to glycine, formaldehyde, and H₂ O₂ in vitro. AtSOX also attacked other N-methyl amino acids and, like mammalian SOXs, catalyzed the oxidation of L-pipecolate to ¹-piperideine-6-carboxylate. Like bacterial monomeric SOXs, AtSOX was active as a monomer, contained FAD covalently bound to a cysteine residue near the C terminus, and was not stimulated by tetrahydrofolate. Although AtSOX lacks a typical peroxisome-targeting signal, in vitro assays established that it is imported into peroxisomes. Quantitation of mRNA showed that AtSOX is expressed at a low level throughout the plant and is not sarcosine-inducible. Consistent with a low level of AtSOX expression, Arabidopsis plantlets slowly metabolized supplied [¹⁴C]sarcosine to glycine and serine. Gas chromatography-mass spectrometry analysis revealed low levels of pipecolate but almost no sarcosine in wild type Arabidopsis and showed that pipecolate but not sarcosine accumulated 6-fold when AtSOX expression was suppressed by RNA interference. Moreover, the pipecolate catabolite -aminoadipate decreased 30-fold in RNA interference plants. These data indicate that pipecolate is the endogenous substrate for SOX in plants and that plants can utilize exogenous sarcosine opportunistically, sarcosine being a common soil metabolite.

Received for publication, January 5, 2004 , and in revised form, February 6, 2004.

^* This work was supported in part by the Florida Agricultural Experimental Station, by an endowment from the C. V. Griffin, Sr. Foundation, and by Grants MCB-0114117 and MCB-0327241 from the National Science Foundation and approved for publication as Journal Series number R.09954. 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.

^** To whom correspondence should be addressed: Horticultural Sciences Dept., University of Florida, P. O. Box 110690, Gainesville, Florida 32611. Tel.: 352-392-1928 (ext. 334); Fax: 352-392-5653; E-mail: adha{at}mail.ifas.ufl.edu.

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