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) synt hetase (PS) by functional complementation of an Escherichia coli pan C muta nt (AT1371), A rice (Oryza sativum) expressed sequence tag, identified by s equence 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 e xtensions 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 ther efore likely to be cytosolic, Southern analysis of Lotus genomic DNA indica tes that there is a single gene for PS. Recombinant PS from Lotus, overexpr essed in E, coli AT1371, is a dimer, The enzyme requires D-pantoate, beta-a lanine and ATP for activity and has a higher affinity for pantoate (K-m 45 mu M) than for beta-alanine (K-m 990 mu M). Uncompetitive substrate inhibit ion becomes significant at pantoate concentrations above 1 mM. The enzyme d isplays optimal activity at about 0.5 mM pantoate (k(cat) 0.63 s(-1)) and a t pH 7.8. Neither oxopantoate nor pantoyl-lactone can replace pantoate as s ubstrate. Antibodies raised against recombinant PS detected a band of 34 kD a in Western blots of Lotus proteins from both roots and leaves, The implic ations of these findings for pantothenate biosynthesis in plants are discus sed.