Metabolism of the folate precursor p-aminobenzoate in plants - Glucose ester formation and vacuolar storage

TY - JOUR

T1 - Metabolism of the folate precursor p-aminobenzoate in plants - Glucose ester formation and vacuolar storage

AU - Eudes, Aymerick

AU - Bozzo, Gale G.

AU - Waller, Jeffrey C.

AU - Naponelli, Valeria

AU - Lim, Eng-Kiat

AU - Bowles, Dianna J.

AU - Gregory, Jesse F.

AU - Hanson, Andrew D.

PY - 2008/5/30

Y1 - 2008/5/30

N2 - 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 [C-14]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 [C-14]pABA-fed pea leaves were estimated to contain >= 88% of the [C-14]pABA-Glc formed. In total, these data and the fact that isolated mitochondria did not take up [H-3]pABA-Glc, suggest that the glucose ester represents a storage form of pABA that does not contribute directly to folate synthesis.

AB - 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 [C-14]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 [C-14]pABA-fed pea leaves were estimated to contain >= 88% of the [C-14]pABA-Glc formed. In total, these data and the fact that isolated mitochondria did not take up [H-3]pABA-Glc, suggest that the glucose ester represents a storage form of pABA that does not contribute directly to folate synthesis.

KW - CELL-SUSPENSION-CULTURES

KW - PHENOLIC-COMPOUNDS

KW - ORGANIC-ACIDS

KW - COMPARTMENTATION

KW - MITOCHONDRIA

KW - NUCLEOTIDES

KW - SYNTHASE

KW - BINDING

KW - GLYCOSYLTRANSFERASES

KW - BIOSYNTHESIS

UR - http://www.scopus.com/inward/record.url?scp=47249152852&partnerID=8YFLogxK

U2 - 10.1074/jbc.M709591200

DO - 10.1074/jbc.M709591200

M3 - Article

VL - 283

SP - 15451

EP - 15459

JO - The Journal of biological chemistry

JF - The Journal of biological chemistry

SN - 0021-9258

IS - 22

ER -