Organisation of the pantothenate (vitamin B5) biosynthesis pathway in higher plants.
Pantothenate (vitamin B5) is the precursor for the biosynthesis of the phosphopantetheine moiety of coenzyme A and acyl carrier protein, and is synthesised in Escherichia coli by four enzymic reactions. Ketopantoate hydroxymethyltransferase (KPHMT) and pantothenate synthetase (PtS) catalyse the first and last steps, respectively. Two genes encoding KPHMT and one for PtS were identified in the Arabidopsis thaliana genome, and cDNAs for all three genes were amplified by PCR. The cDNAs were able to complement their respective E. coli auxotrophs, demonstrating that they encoded functional enzymes. Subcellular localisation of the proteins was investigated using green fluorescent protein (GFP) fusions and confocal microscopy. The two KPHMT-GFP fusion proteins were targeted exclusively to mitochondria, whereas PtS-GFP was found in the cytosol. This implies that there must be transporters for pathway intermediates. KPHMT enzyme activity could be measured in purified mitochondria from both pea leaves and Arabidopsis suspension cultures. We investigated whether Arabidopsis encoded homologues of the remaining two pantothenate biosynthesis enzymes from E. coli, l-aspartate-alpha-decarboxylase (ADC) and ketopantoate reductase (KPR). No homologue of ADC could be identified using either conventional blast or searches with the program fugue in which the structure of the E. coli ADC was compared to all the annotated proteins in Arabidopsis. ADC also appears to be absent from the genome of the yeast, Saccharomyces cerevisiae, by the same criteria. In contrast, a putative Arabidopsis oxidoreductase with some similarity to KPR was identified with fugue.
Ottenhof
Harald H
HH
Department of Plant Sciences, Downing Street, Cambridge CB2 3EA, UK.
Ashurst
Jennifer L
JL
Whitney
Heather M
HM
Saldanha
S Adrian
SA
Schmitzberger
Florian
F
Gweon
Hyun Soon
HS
Blundell
Tom L
TL
Abell
Chris
C
Smith
Alison G
AG
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000737:chemistry / Q000201:enzymology / Q000235:genetics*; D029681:Arabidopsis Proteins / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D003600:Cytosol / Q000378:metabolism; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D049452:Green Fluorescent Proteins; D019877:Hydroxymethyl and Formyl Transferases / Q000235:genetics* / Q000378:metabolism*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008928:Mitochondria / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010205:Pantothenic Acid / Q000096:biosynthesis*; D010453:Peptide Synthases / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D029681:Arabidopsis Proteins; D018076:DNA, Complementary; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D010205:Pantothenic Acid; D019877:Hydroxymethyl and Formyl Transferases; C021477:3-methyl-2-oxobutanoate hydroxymethyltransferase; D010453:Peptide Synthases; C022318:pantothenate synthetase