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J. Biol. Chem., Vol. 283, Issue 22, 15451-15459, May 30, 2008

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Metabolism of the Folate Precursor p-Aminobenzoate in Plants

GLUCOSE ESTER FORMATION AND VACUOLAR STORAGE^*

Aymerick Eudes¹, Gale G. Bozzo¹, Jeffrey C. Waller, Valeria Naponelli, Eng-Kiat Lim^¶, Dianna J. Bowles^¶, Jesse F. Gregory, III, and Andrew D. Hanson²

From the Departments of Horticultural Sciences and Food Science and Human Nutrition, University of Florida, Gainesville, Florida 32611 and the ^¶Centre for Novel Agricultural Products, Department of Biology, University of York, York YO10 5DD, United Kingdom

Plants produce p-aminobenzoate (pABA) in chloroplasts and use it for folate synthesis in mitochondria. In plant tissues, however, pABA is known to occur predominantly as its glucose ester (pABA-Glc), and the role of this metabolite in folate synthesis has not been defined. In this study, the UDP-glucose:pABA acyl-glucosyltransferase (pAGT) activity in Arabidopsis extracts was found to reside principally (95%) in one isoform with an apparent K_m for pABA of 0.12 mM. Screening of recombinant Arabidopsis UDP-glycosyltransferases identified only three that recognized pABA. One of these (UGT75B1) exhibited a far higher k_cat/K_m value than the others and a far lower apparent K_m for pABA (0.12 mM), suggesting its identity with the principal enzyme in vivo. Supporting this possibility, ablation of UGT75B1 reduced extractable pAGT activity by 95%, in vivo [¹⁴C]pABA glucosylation by 77%, and the endogenous pABA-Glc/pABA ratio by 9-fold. The K_eq for the pABA esterification reaction was found to be 3 x 10^-3. Taken with literature data on the cytosolic location of pAGT activity and on cytosolic UDP-glucose/UDP ratios, this K_eq value allowed estimation that only 4% of cytosolic pABA is esterified. That pABA-Glc predominates in planta therefore implies that it is sequestered away from the cytosol and, consistent with this possibility, vacuoles isolated from [¹⁴C]pABA-fed pea leaves were estimated to contain88% of the [¹⁴C]pABA-Glc formed. In total, these data and the fact that isolated mitochondria did not take up [³H]pABA-Glc, suggest that the glucose ester represents a storage form of pABA that does not contribute directly to folate synthesis.

Received for publication, November 26, 2007 , and in revised form, March 7, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grant R01 GM071382. This work was also supported by an endowment from the C. V. Griffin, Sr. Foundation. 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.

¹ These authors contributed equally to this work.

² To whom correspondence should be addressed: University of Florida, Horticultural Sciences Dept., P.O. Box 110690, Gainesville, FL 32611-0690. Fax: 352-392-5653; E-mail: adha{at}ufl.edu.

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