Abstract/Summary

Pantothenate (vitamin B5) is the precursor for the biosynthesis of the phosphopantetheine moiety of coenzymeA and acyl carrier protein, and is synthesised in Esche ri chia col i by four enzymic reactions. Ketopantoatehydroxymethyltransferase (KPHMT) and pantothenate synthetase (PtS) catalyse the ®rst and last steps, respec-tively. Two genes encoding KPHMT and one for PtS were identi®ed in the Arabidopsis thaliana genome, andcDNAs for all three genes were ampli®ed by PCR. The cDNAs were able to complement their respective E. coliauxotrophs, demonstrating that they encoded functional enzymes. Subcellular localisation of the proteins wasinvestigated using green ¯uorescent protein (GFP) fusions and confocal microscopy. The two KPHMT±GFPfusion proteins were targeted exclusively to mitochondria, whereas PtS±GFP was found in the cytosol. Thisimplies that there must be transporters for pathway intermediates. KPHMT enzyme activity could bemeasured in puri®ed mitochondria from both pea leaves and Ar ab ido ps is suspension cultures. We investigatedwhether Arabidopsis encoded homologues of the remaining two pantothenate biosynthesis enzymes fromE. coli,L-aspartate-a-decarboxylase (ADC) and ketopantoate reductase (KPR). No homologue of ADCcould be identi®ed using either conventionalBLASTor searches with the programFUGUEin which thestructure of the E. coli ADC was compared to all the annotated proteins in Arabidopsis. ADC also appearsto be absent from the genome of the yeast, Saccharomyces cerevisiae, by the same criteria. In contrast, aputative Ar ab ido psis oxidoreductase with some similarity to KPR was identi®ed withFUGUE.