Reference "The final step of pantothenate biosynthesis in higher plants: cloning and characterization of pantothenate synthetase from Lotus japonicus and Oryza sativum (rice)"

Reference ID

1117

Title

The final step of pantothenate biosynthesis in higher plants: cloning and characterization of pantothenate synthetase from Lotus japonicus and Oryza sativum (rice)

Source

Biochemical Journal, 1999, vol. 341, pp. 669-678

Authors (4)

Genschel-U	Powell-C-A
Abell-C	Smith-A-G

Abstract

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.