Purification, molecular cloning, and sequence analysis of sucrose-6(F)-phosphate phosphohydrolase from plants

Sucrose-6(F)-phosphate phosphohydrolase (SPP; EC 3.1.3.24) catalyzes the fi nal step in the pathway of sucrose biosynthesis and is the only enzyme of p hotosynthetic carbon assimilation for which the gene has not been identifie d. The enzyme was purified to homogeneity from rice (Oryza sativa L.) leave s and partially sequenced. The rice leaf enzyme is a dimer with a native mo lecular mass of 100 kDa and a subunit molecular mass of 50 kDa, The enzyme is highly specific for sucrose 6(F)-phosphate with a K-m of 65 muM and a sp ecific activity of 1250 mu mol min(-1) mg(-1) protein. The activity is depe ndent on Mg2+ with a remarkably low K-a of 8-9 muM and is weakly inhibited by sucrose, Three peptides from cleavage of the purified rice SPP with endo proteinase Lys-C showed similarity to the deduced amino acid sequences of t hree predicted open reading frames (ORF) in the Arabidopsis thaliana genome and one in the genome of the cyanobacterium Synechocystis sp. PCC6803, as well as cDNA clones from Arabidopsis, maize, and other species in the GenBa nk database of expressed sequence tags. The putative maize SPP cDNA clone c ontained an ORF encoding a 420-amino acid polypeptide. Heterologous express ion in Escherichia coli showed that this cDNA clone encoded a functional SP P enzyme. The 260-amino acid N-terminal catalytic domain of the maize SPP i s homologous to the C-terminal region of sucrose-phosphate synthase, A PSI- BLAST search of the GenBank database indicated that the maize SPP is a memb er of the haloacid dehalogenase hydrolase/phosphatase superfamily.