The final step of pantothenate biosynthesis in higher plants: cloning and characterization of pantothenate synthetase from Lotus japonicus and Oryza sativum (rice).
We have isolated a Lotus japonicus cDNA for pantothenate (vitamin B(5)) synthetase (PS) by functional complementation of an Escherichia coli panC mutant (AT1371). A rice (Oryza sativum) expressed sequence tag, identified by sequence similarity to PS, was also able to complement the E. coli auxotroph, as was an open reading frame from Saccharomyces cerevisiae (baker's yeast). The Lotus and rice cDNAs encode proteins of approx. 34 kDa, which are 65% similar at the amino acid level and do not appear to encode N-terminal extensions by comparison with PS sequences from other organisms. Furthermore, analysis of genomic sequence flanking the coding sequence for PS in Lotus suggests the original cDNA is full-length. The Lotus and rice PSs are therefore likely to be cytosolic. Southern analysis of Lotus genomic DNA indicates that there is a single gene for PS. Recombinant PS from Lotus, overexpressed in E. coli AT1371, is a dimer. The enzyme requires d-pantoate, beta-alanine and ATP for activity and has a higher affinity for pantoate (K(m) 45 microM) than for beta-alanine (K(m) 990 microM). Uncompetitive substrate inhibition becomes significant at pantoate concentrations above 1 mM. The enzyme displays optimal activity at about 0.5 mM pantoate (k(cat) 0.63 s(-1)) and at pH 7.8. Neither oxopantoate nor pantoyl-lactone can replace pantoate as substrate. Antibodies raised against recombinant PS detected a band of 34 kDa in Western blots of Lotus proteins from both roots and leaves. The implications of these findings for pantothenate biosynthesis in plants are discussed.
Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge, CB2 3EA, U.K.
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Genschel
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Powell
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Smith
D000595:Amino Acid Sequence; D001483:Base Sequence; D015139:Blotting, Southern; D015153:Blotting, Western; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics; D005816:Genetic Complementation Test; D007700:Kinetics; D008969:Molecular Sequence Data; D010205:Pantothenic Acid / Q000096:biosynthesis*; D010453:Peptide Synthases / Q000235:genetics* / Q000378:metabolism; D010944:Plants / Q000201:enzymology* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity
D017931:DNA Primers; D011994:Recombinant Proteins; D010205:Pantothenic Acid; D010453:Peptide Synthases; C022318:pantothenate synthetase